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Erwin R Schmidt Institut fuumlr Molekulargenetik
Vorlesung 10 01 07 2014
Thema Gentechnologie
Pyrosequenzierung
The Pyrosequencingtrade technology is a relatively new DNA sequencing method originally developed here at KTH at the Department of Biotechnology The technology has been commercialized and is today marketed by Biotage AB The technique utilizes the cooperativity between four different enzymes and the phenomenon of bioluminescence to monitor the incorporation of nucleotides into the DNA A short description of the steps in the Pyrosequencing process is given below Initial step The reaction mixture consists of the four enzymes (DNA polymerase ATP sulfurylase luciferase and apyrase) different substrates needed for the reactions and the single stranded DNA to be sequenced Step 1 - Polymerase One of the four nucleotides dNTP (dATP dCTP dGTP dTTP) is added to the reaction mixture If the added nucleotide is complementary to the base in the DNA strand it is incorporated and inorganic pyrophosphate (PPi) is released Step 2 - ATP sulfurylase The PPi is converted into ATP by the enzyme ATP sulfurylase Step 3 - Luciferase The luciferase catalyzes a reaction where ATP is used to generate light The amount of light is proportional to the amount of ATP and hence also proportional to the amount of incorporated nucleotides via the PPi The light is then detected by a CCD camera Step 4 - Apyrase Remaining dNTP and ATP are degraded by the apyrase before the next nucleotide in the iterative cycle is added to the reaction mixture My research is devoted to developing a good mathematical model of the reaction system This will help us to understand the mechanisms governing the system in detail Once a satisfactory model has been developed it can be used to optimize the method with respect to substrate and enzyme concentrations as well as the choice of enzymes (kinetic parameters) As the demand for even better DNA sequencing techniques is steadily increasing as new applications arise there is a lot to gain by optimization
bdquoNext Generationldquo Sequencing (NGS)
Erwin R Schmidt Institut fuumlr Molekulargenetik
Johannes Gutenberg Universitaumlt Mainz
DNA-Sequencing
A brief historical overview The different platforms of NGS Benchtop versus High Output Cost and Reliabilty Future technologies Summary
NGS Short History of (Nucleotide) Sequencing How many generations do we have
First Generation Sequencing
bull Nearest neighbor technology
bull Combined with sequence or base specific nuclease digestion
The first nucleotide sequence of a complete biomolecule was the Alanine tRNA of Yeast
by Robert W Holley et al in 1964 Nobel Prize in Physiology and Medicine 1968
(for 77 nt)
Generation 2 The real breakthrough
bull 1975 -1977 the bdquoSanger Sequencingldquo ndash sequencing by synthesis
bull Nobel Prize Chemistry 1980
bull 1977 the bdquoMaxam and Gilbertldquo Sequencing - sequencing by chemical degradation
bull Nobel Prize Chemistry 1980
Development of the Sanger Sequencing
1975 Sanger and Coulson published the +- method Sanger F Coulson AR A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase J Mol Biol 1975 May 2594(3)441ndash448
1977 Sanger Nicklen and Coulson published the chain terminator method F Sanger S Nicklen and A R Coulson DNA sequencing with chain-terminating inhibitors Proc Natl Acad Sci U S Dec 1977 Av74(12) 5463-5467
The Maxam and Gilbert Method based on base specific chemical degradation of end-
labelled DNA-restriction fragments
In the same year (1977) but 10 months before Sanger published the chain terminator Method Maxam and Gilbert published their DNA sequencing method based on chemical degradation of end-labelled DNA restriction fragments A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Maxam and Gilbert sequencing
bull A very robust method
bull Not sensitive to secondary structures
bull Shows base modification
But
bull Requires work with with
bull strong carcinogens and miliCuries of
radioisotopes
bull Is not automatable
bull Very laborious and requires long
exposition times
A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Sanger sequencing
bull Reading the sequence easier
bull No carcinogenic chemicals involved
bull Exposure times were only a few hours
bull the sequencing reactions could be done by
acute the technician
bull but
the natural DNA-Polymerases are sensitive to
secondary structures and stretches of
homopolymeric nucleotides This changed
only when the bdquosequenasesldquo were invented
Sanger-sequencing has won the race
Maxam and Gilbert
Number of citations
- 7690 times
Sanger Nicklen and Coulson
Number of citations
ndash 62757 times
Source Google Scholar
Generation 3 on line sequencing
- number of different techniques
- all based on fluorescently labelled DNA framents which could be detected and tranferred automatically to a computer
- automated bdquobase callingldquo
bdquoClassicalldquo on line sequencing is still in use
bull The demand is still increasing
bull Results are robust low error rate lt 11000-110000 bp
bull Up to 1500 nt readable in a row
bull Cost per sample ~ 3-5 euro (014 Cent Bpds)
bull Comprehensive service available commercially
Generation 4
Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod
of the yearldquo
bull introduces a new dimension in sequence determination
bull Several platforms exist providing different possibilities
The advent of NGS is reflected by the number of genome projects and data base entries
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
In particular bacterial genome projects boost since 2008
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24
Source httpgenomesonlineorgcgi-binGOLDindexcgi
Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi
Incomplete Genome Projects 27988 Archaeal 457
Bacterial 19494 Eukaryal 6413
Last updated 2014-01-24
Source GOLD = Genomes Online Database at the DOE Joint Genome Institute
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Pyrosequenzierung
The Pyrosequencingtrade technology is a relatively new DNA sequencing method originally developed here at KTH at the Department of Biotechnology The technology has been commercialized and is today marketed by Biotage AB The technique utilizes the cooperativity between four different enzymes and the phenomenon of bioluminescence to monitor the incorporation of nucleotides into the DNA A short description of the steps in the Pyrosequencing process is given below Initial step The reaction mixture consists of the four enzymes (DNA polymerase ATP sulfurylase luciferase and apyrase) different substrates needed for the reactions and the single stranded DNA to be sequenced Step 1 - Polymerase One of the four nucleotides dNTP (dATP dCTP dGTP dTTP) is added to the reaction mixture If the added nucleotide is complementary to the base in the DNA strand it is incorporated and inorganic pyrophosphate (PPi) is released Step 2 - ATP sulfurylase The PPi is converted into ATP by the enzyme ATP sulfurylase Step 3 - Luciferase The luciferase catalyzes a reaction where ATP is used to generate light The amount of light is proportional to the amount of ATP and hence also proportional to the amount of incorporated nucleotides via the PPi The light is then detected by a CCD camera Step 4 - Apyrase Remaining dNTP and ATP are degraded by the apyrase before the next nucleotide in the iterative cycle is added to the reaction mixture My research is devoted to developing a good mathematical model of the reaction system This will help us to understand the mechanisms governing the system in detail Once a satisfactory model has been developed it can be used to optimize the method with respect to substrate and enzyme concentrations as well as the choice of enzymes (kinetic parameters) As the demand for even better DNA sequencing techniques is steadily increasing as new applications arise there is a lot to gain by optimization
bdquoNext Generationldquo Sequencing (NGS)
Erwin R Schmidt Institut fuumlr Molekulargenetik
Johannes Gutenberg Universitaumlt Mainz
DNA-Sequencing
A brief historical overview The different platforms of NGS Benchtop versus High Output Cost and Reliabilty Future technologies Summary
NGS Short History of (Nucleotide) Sequencing How many generations do we have
First Generation Sequencing
bull Nearest neighbor technology
bull Combined with sequence or base specific nuclease digestion
The first nucleotide sequence of a complete biomolecule was the Alanine tRNA of Yeast
by Robert W Holley et al in 1964 Nobel Prize in Physiology and Medicine 1968
(for 77 nt)
Generation 2 The real breakthrough
bull 1975 -1977 the bdquoSanger Sequencingldquo ndash sequencing by synthesis
bull Nobel Prize Chemistry 1980
bull 1977 the bdquoMaxam and Gilbertldquo Sequencing - sequencing by chemical degradation
bull Nobel Prize Chemistry 1980
Development of the Sanger Sequencing
1975 Sanger and Coulson published the +- method Sanger F Coulson AR A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase J Mol Biol 1975 May 2594(3)441ndash448
1977 Sanger Nicklen and Coulson published the chain terminator method F Sanger S Nicklen and A R Coulson DNA sequencing with chain-terminating inhibitors Proc Natl Acad Sci U S Dec 1977 Av74(12) 5463-5467
The Maxam and Gilbert Method based on base specific chemical degradation of end-
labelled DNA-restriction fragments
In the same year (1977) but 10 months before Sanger published the chain terminator Method Maxam and Gilbert published their DNA sequencing method based on chemical degradation of end-labelled DNA restriction fragments A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Maxam and Gilbert sequencing
bull A very robust method
bull Not sensitive to secondary structures
bull Shows base modification
But
bull Requires work with with
bull strong carcinogens and miliCuries of
radioisotopes
bull Is not automatable
bull Very laborious and requires long
exposition times
A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Sanger sequencing
bull Reading the sequence easier
bull No carcinogenic chemicals involved
bull Exposure times were only a few hours
bull the sequencing reactions could be done by
acute the technician
bull but
the natural DNA-Polymerases are sensitive to
secondary structures and stretches of
homopolymeric nucleotides This changed
only when the bdquosequenasesldquo were invented
Sanger-sequencing has won the race
Maxam and Gilbert
Number of citations
- 7690 times
Sanger Nicklen and Coulson
Number of citations
ndash 62757 times
Source Google Scholar
Generation 3 on line sequencing
- number of different techniques
- all based on fluorescently labelled DNA framents which could be detected and tranferred automatically to a computer
- automated bdquobase callingldquo
bdquoClassicalldquo on line sequencing is still in use
bull The demand is still increasing
bull Results are robust low error rate lt 11000-110000 bp
bull Up to 1500 nt readable in a row
bull Cost per sample ~ 3-5 euro (014 Cent Bpds)
bull Comprehensive service available commercially
Generation 4
Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod
of the yearldquo
bull introduces a new dimension in sequence determination
bull Several platforms exist providing different possibilities
The advent of NGS is reflected by the number of genome projects and data base entries
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
In particular bacterial genome projects boost since 2008
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24
Source httpgenomesonlineorgcgi-binGOLDindexcgi
Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi
Incomplete Genome Projects 27988 Archaeal 457
Bacterial 19494 Eukaryal 6413
Last updated 2014-01-24
Source GOLD = Genomes Online Database at the DOE Joint Genome Institute
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
The Pyrosequencingtrade technology is a relatively new DNA sequencing method originally developed here at KTH at the Department of Biotechnology The technology has been commercialized and is today marketed by Biotage AB The technique utilizes the cooperativity between four different enzymes and the phenomenon of bioluminescence to monitor the incorporation of nucleotides into the DNA A short description of the steps in the Pyrosequencing process is given below Initial step The reaction mixture consists of the four enzymes (DNA polymerase ATP sulfurylase luciferase and apyrase) different substrates needed for the reactions and the single stranded DNA to be sequenced Step 1 - Polymerase One of the four nucleotides dNTP (dATP dCTP dGTP dTTP) is added to the reaction mixture If the added nucleotide is complementary to the base in the DNA strand it is incorporated and inorganic pyrophosphate (PPi) is released Step 2 - ATP sulfurylase The PPi is converted into ATP by the enzyme ATP sulfurylase Step 3 - Luciferase The luciferase catalyzes a reaction where ATP is used to generate light The amount of light is proportional to the amount of ATP and hence also proportional to the amount of incorporated nucleotides via the PPi The light is then detected by a CCD camera Step 4 - Apyrase Remaining dNTP and ATP are degraded by the apyrase before the next nucleotide in the iterative cycle is added to the reaction mixture My research is devoted to developing a good mathematical model of the reaction system This will help us to understand the mechanisms governing the system in detail Once a satisfactory model has been developed it can be used to optimize the method with respect to substrate and enzyme concentrations as well as the choice of enzymes (kinetic parameters) As the demand for even better DNA sequencing techniques is steadily increasing as new applications arise there is a lot to gain by optimization
bdquoNext Generationldquo Sequencing (NGS)
Erwin R Schmidt Institut fuumlr Molekulargenetik
Johannes Gutenberg Universitaumlt Mainz
DNA-Sequencing
A brief historical overview The different platforms of NGS Benchtop versus High Output Cost and Reliabilty Future technologies Summary
NGS Short History of (Nucleotide) Sequencing How many generations do we have
First Generation Sequencing
bull Nearest neighbor technology
bull Combined with sequence or base specific nuclease digestion
The first nucleotide sequence of a complete biomolecule was the Alanine tRNA of Yeast
by Robert W Holley et al in 1964 Nobel Prize in Physiology and Medicine 1968
(for 77 nt)
Generation 2 The real breakthrough
bull 1975 -1977 the bdquoSanger Sequencingldquo ndash sequencing by synthesis
bull Nobel Prize Chemistry 1980
bull 1977 the bdquoMaxam and Gilbertldquo Sequencing - sequencing by chemical degradation
bull Nobel Prize Chemistry 1980
Development of the Sanger Sequencing
1975 Sanger and Coulson published the +- method Sanger F Coulson AR A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase J Mol Biol 1975 May 2594(3)441ndash448
1977 Sanger Nicklen and Coulson published the chain terminator method F Sanger S Nicklen and A R Coulson DNA sequencing with chain-terminating inhibitors Proc Natl Acad Sci U S Dec 1977 Av74(12) 5463-5467
The Maxam and Gilbert Method based on base specific chemical degradation of end-
labelled DNA-restriction fragments
In the same year (1977) but 10 months before Sanger published the chain terminator Method Maxam and Gilbert published their DNA sequencing method based on chemical degradation of end-labelled DNA restriction fragments A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Maxam and Gilbert sequencing
bull A very robust method
bull Not sensitive to secondary structures
bull Shows base modification
But
bull Requires work with with
bull strong carcinogens and miliCuries of
radioisotopes
bull Is not automatable
bull Very laborious and requires long
exposition times
A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Sanger sequencing
bull Reading the sequence easier
bull No carcinogenic chemicals involved
bull Exposure times were only a few hours
bull the sequencing reactions could be done by
acute the technician
bull but
the natural DNA-Polymerases are sensitive to
secondary structures and stretches of
homopolymeric nucleotides This changed
only when the bdquosequenasesldquo were invented
Sanger-sequencing has won the race
Maxam and Gilbert
Number of citations
- 7690 times
Sanger Nicklen and Coulson
Number of citations
ndash 62757 times
Source Google Scholar
Generation 3 on line sequencing
- number of different techniques
- all based on fluorescently labelled DNA framents which could be detected and tranferred automatically to a computer
- automated bdquobase callingldquo
bdquoClassicalldquo on line sequencing is still in use
bull The demand is still increasing
bull Results are robust low error rate lt 11000-110000 bp
bull Up to 1500 nt readable in a row
bull Cost per sample ~ 3-5 euro (014 Cent Bpds)
bull Comprehensive service available commercially
Generation 4
Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod
of the yearldquo
bull introduces a new dimension in sequence determination
bull Several platforms exist providing different possibilities
The advent of NGS is reflected by the number of genome projects and data base entries
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
In particular bacterial genome projects boost since 2008
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24
Source httpgenomesonlineorgcgi-binGOLDindexcgi
Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi
Incomplete Genome Projects 27988 Archaeal 457
Bacterial 19494 Eukaryal 6413
Last updated 2014-01-24
Source GOLD = Genomes Online Database at the DOE Joint Genome Institute
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
bdquoNext Generationldquo Sequencing (NGS)
Erwin R Schmidt Institut fuumlr Molekulargenetik
Johannes Gutenberg Universitaumlt Mainz
DNA-Sequencing
A brief historical overview The different platforms of NGS Benchtop versus High Output Cost and Reliabilty Future technologies Summary
NGS Short History of (Nucleotide) Sequencing How many generations do we have
First Generation Sequencing
bull Nearest neighbor technology
bull Combined with sequence or base specific nuclease digestion
The first nucleotide sequence of a complete biomolecule was the Alanine tRNA of Yeast
by Robert W Holley et al in 1964 Nobel Prize in Physiology and Medicine 1968
(for 77 nt)
Generation 2 The real breakthrough
bull 1975 -1977 the bdquoSanger Sequencingldquo ndash sequencing by synthesis
bull Nobel Prize Chemistry 1980
bull 1977 the bdquoMaxam and Gilbertldquo Sequencing - sequencing by chemical degradation
bull Nobel Prize Chemistry 1980
Development of the Sanger Sequencing
1975 Sanger and Coulson published the +- method Sanger F Coulson AR A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase J Mol Biol 1975 May 2594(3)441ndash448
1977 Sanger Nicklen and Coulson published the chain terminator method F Sanger S Nicklen and A R Coulson DNA sequencing with chain-terminating inhibitors Proc Natl Acad Sci U S Dec 1977 Av74(12) 5463-5467
The Maxam and Gilbert Method based on base specific chemical degradation of end-
labelled DNA-restriction fragments
In the same year (1977) but 10 months before Sanger published the chain terminator Method Maxam and Gilbert published their DNA sequencing method based on chemical degradation of end-labelled DNA restriction fragments A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Maxam and Gilbert sequencing
bull A very robust method
bull Not sensitive to secondary structures
bull Shows base modification
But
bull Requires work with with
bull strong carcinogens and miliCuries of
radioisotopes
bull Is not automatable
bull Very laborious and requires long
exposition times
A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Sanger sequencing
bull Reading the sequence easier
bull No carcinogenic chemicals involved
bull Exposure times were only a few hours
bull the sequencing reactions could be done by
acute the technician
bull but
the natural DNA-Polymerases are sensitive to
secondary structures and stretches of
homopolymeric nucleotides This changed
only when the bdquosequenasesldquo were invented
Sanger-sequencing has won the race
Maxam and Gilbert
Number of citations
- 7690 times
Sanger Nicklen and Coulson
Number of citations
ndash 62757 times
Source Google Scholar
Generation 3 on line sequencing
- number of different techniques
- all based on fluorescently labelled DNA framents which could be detected and tranferred automatically to a computer
- automated bdquobase callingldquo
bdquoClassicalldquo on line sequencing is still in use
bull The demand is still increasing
bull Results are robust low error rate lt 11000-110000 bp
bull Up to 1500 nt readable in a row
bull Cost per sample ~ 3-5 euro (014 Cent Bpds)
bull Comprehensive service available commercially
Generation 4
Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod
of the yearldquo
bull introduces a new dimension in sequence determination
bull Several platforms exist providing different possibilities
The advent of NGS is reflected by the number of genome projects and data base entries
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
In particular bacterial genome projects boost since 2008
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24
Source httpgenomesonlineorgcgi-binGOLDindexcgi
Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi
Incomplete Genome Projects 27988 Archaeal 457
Bacterial 19494 Eukaryal 6413
Last updated 2014-01-24
Source GOLD = Genomes Online Database at the DOE Joint Genome Institute
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
DNA-Sequencing
A brief historical overview The different platforms of NGS Benchtop versus High Output Cost and Reliabilty Future technologies Summary
NGS Short History of (Nucleotide) Sequencing How many generations do we have
First Generation Sequencing
bull Nearest neighbor technology
bull Combined with sequence or base specific nuclease digestion
The first nucleotide sequence of a complete biomolecule was the Alanine tRNA of Yeast
by Robert W Holley et al in 1964 Nobel Prize in Physiology and Medicine 1968
(for 77 nt)
Generation 2 The real breakthrough
bull 1975 -1977 the bdquoSanger Sequencingldquo ndash sequencing by synthesis
bull Nobel Prize Chemistry 1980
bull 1977 the bdquoMaxam and Gilbertldquo Sequencing - sequencing by chemical degradation
bull Nobel Prize Chemistry 1980
Development of the Sanger Sequencing
1975 Sanger and Coulson published the +- method Sanger F Coulson AR A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase J Mol Biol 1975 May 2594(3)441ndash448
1977 Sanger Nicklen and Coulson published the chain terminator method F Sanger S Nicklen and A R Coulson DNA sequencing with chain-terminating inhibitors Proc Natl Acad Sci U S Dec 1977 Av74(12) 5463-5467
The Maxam and Gilbert Method based on base specific chemical degradation of end-
labelled DNA-restriction fragments
In the same year (1977) but 10 months before Sanger published the chain terminator Method Maxam and Gilbert published their DNA sequencing method based on chemical degradation of end-labelled DNA restriction fragments A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Maxam and Gilbert sequencing
bull A very robust method
bull Not sensitive to secondary structures
bull Shows base modification
But
bull Requires work with with
bull strong carcinogens and miliCuries of
radioisotopes
bull Is not automatable
bull Very laborious and requires long
exposition times
A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Sanger sequencing
bull Reading the sequence easier
bull No carcinogenic chemicals involved
bull Exposure times were only a few hours
bull the sequencing reactions could be done by
acute the technician
bull but
the natural DNA-Polymerases are sensitive to
secondary structures and stretches of
homopolymeric nucleotides This changed
only when the bdquosequenasesldquo were invented
Sanger-sequencing has won the race
Maxam and Gilbert
Number of citations
- 7690 times
Sanger Nicklen and Coulson
Number of citations
ndash 62757 times
Source Google Scholar
Generation 3 on line sequencing
- number of different techniques
- all based on fluorescently labelled DNA framents which could be detected and tranferred automatically to a computer
- automated bdquobase callingldquo
bdquoClassicalldquo on line sequencing is still in use
bull The demand is still increasing
bull Results are robust low error rate lt 11000-110000 bp
bull Up to 1500 nt readable in a row
bull Cost per sample ~ 3-5 euro (014 Cent Bpds)
bull Comprehensive service available commercially
Generation 4
Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod
of the yearldquo
bull introduces a new dimension in sequence determination
bull Several platforms exist providing different possibilities
The advent of NGS is reflected by the number of genome projects and data base entries
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
In particular bacterial genome projects boost since 2008
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24
Source httpgenomesonlineorgcgi-binGOLDindexcgi
Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi
Incomplete Genome Projects 27988 Archaeal 457
Bacterial 19494 Eukaryal 6413
Last updated 2014-01-24
Source GOLD = Genomes Online Database at the DOE Joint Genome Institute
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
NGS Short History of (Nucleotide) Sequencing How many generations do we have
First Generation Sequencing
bull Nearest neighbor technology
bull Combined with sequence or base specific nuclease digestion
The first nucleotide sequence of a complete biomolecule was the Alanine tRNA of Yeast
by Robert W Holley et al in 1964 Nobel Prize in Physiology and Medicine 1968
(for 77 nt)
Generation 2 The real breakthrough
bull 1975 -1977 the bdquoSanger Sequencingldquo ndash sequencing by synthesis
bull Nobel Prize Chemistry 1980
bull 1977 the bdquoMaxam and Gilbertldquo Sequencing - sequencing by chemical degradation
bull Nobel Prize Chemistry 1980
Development of the Sanger Sequencing
1975 Sanger and Coulson published the +- method Sanger F Coulson AR A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase J Mol Biol 1975 May 2594(3)441ndash448
1977 Sanger Nicklen and Coulson published the chain terminator method F Sanger S Nicklen and A R Coulson DNA sequencing with chain-terminating inhibitors Proc Natl Acad Sci U S Dec 1977 Av74(12) 5463-5467
The Maxam and Gilbert Method based on base specific chemical degradation of end-
labelled DNA-restriction fragments
In the same year (1977) but 10 months before Sanger published the chain terminator Method Maxam and Gilbert published their DNA sequencing method based on chemical degradation of end-labelled DNA restriction fragments A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Maxam and Gilbert sequencing
bull A very robust method
bull Not sensitive to secondary structures
bull Shows base modification
But
bull Requires work with with
bull strong carcinogens and miliCuries of
radioisotopes
bull Is not automatable
bull Very laborious and requires long
exposition times
A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Sanger sequencing
bull Reading the sequence easier
bull No carcinogenic chemicals involved
bull Exposure times were only a few hours
bull the sequencing reactions could be done by
acute the technician
bull but
the natural DNA-Polymerases are sensitive to
secondary structures and stretches of
homopolymeric nucleotides This changed
only when the bdquosequenasesldquo were invented
Sanger-sequencing has won the race
Maxam and Gilbert
Number of citations
- 7690 times
Sanger Nicklen and Coulson
Number of citations
ndash 62757 times
Source Google Scholar
Generation 3 on line sequencing
- number of different techniques
- all based on fluorescently labelled DNA framents which could be detected and tranferred automatically to a computer
- automated bdquobase callingldquo
bdquoClassicalldquo on line sequencing is still in use
bull The demand is still increasing
bull Results are robust low error rate lt 11000-110000 bp
bull Up to 1500 nt readable in a row
bull Cost per sample ~ 3-5 euro (014 Cent Bpds)
bull Comprehensive service available commercially
Generation 4
Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod
of the yearldquo
bull introduces a new dimension in sequence determination
bull Several platforms exist providing different possibilities
The advent of NGS is reflected by the number of genome projects and data base entries
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
In particular bacterial genome projects boost since 2008
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24
Source httpgenomesonlineorgcgi-binGOLDindexcgi
Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi
Incomplete Genome Projects 27988 Archaeal 457
Bacterial 19494 Eukaryal 6413
Last updated 2014-01-24
Source GOLD = Genomes Online Database at the DOE Joint Genome Institute
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
First Generation Sequencing
bull Nearest neighbor technology
bull Combined with sequence or base specific nuclease digestion
The first nucleotide sequence of a complete biomolecule was the Alanine tRNA of Yeast
by Robert W Holley et al in 1964 Nobel Prize in Physiology and Medicine 1968
(for 77 nt)
Generation 2 The real breakthrough
bull 1975 -1977 the bdquoSanger Sequencingldquo ndash sequencing by synthesis
bull Nobel Prize Chemistry 1980
bull 1977 the bdquoMaxam and Gilbertldquo Sequencing - sequencing by chemical degradation
bull Nobel Prize Chemistry 1980
Development of the Sanger Sequencing
1975 Sanger and Coulson published the +- method Sanger F Coulson AR A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase J Mol Biol 1975 May 2594(3)441ndash448
1977 Sanger Nicklen and Coulson published the chain terminator method F Sanger S Nicklen and A R Coulson DNA sequencing with chain-terminating inhibitors Proc Natl Acad Sci U S Dec 1977 Av74(12) 5463-5467
The Maxam and Gilbert Method based on base specific chemical degradation of end-
labelled DNA-restriction fragments
In the same year (1977) but 10 months before Sanger published the chain terminator Method Maxam and Gilbert published their DNA sequencing method based on chemical degradation of end-labelled DNA restriction fragments A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Maxam and Gilbert sequencing
bull A very robust method
bull Not sensitive to secondary structures
bull Shows base modification
But
bull Requires work with with
bull strong carcinogens and miliCuries of
radioisotopes
bull Is not automatable
bull Very laborious and requires long
exposition times
A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Sanger sequencing
bull Reading the sequence easier
bull No carcinogenic chemicals involved
bull Exposure times were only a few hours
bull the sequencing reactions could be done by
acute the technician
bull but
the natural DNA-Polymerases are sensitive to
secondary structures and stretches of
homopolymeric nucleotides This changed
only when the bdquosequenasesldquo were invented
Sanger-sequencing has won the race
Maxam and Gilbert
Number of citations
- 7690 times
Sanger Nicklen and Coulson
Number of citations
ndash 62757 times
Source Google Scholar
Generation 3 on line sequencing
- number of different techniques
- all based on fluorescently labelled DNA framents which could be detected and tranferred automatically to a computer
- automated bdquobase callingldquo
bdquoClassicalldquo on line sequencing is still in use
bull The demand is still increasing
bull Results are robust low error rate lt 11000-110000 bp
bull Up to 1500 nt readable in a row
bull Cost per sample ~ 3-5 euro (014 Cent Bpds)
bull Comprehensive service available commercially
Generation 4
Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod
of the yearldquo
bull introduces a new dimension in sequence determination
bull Several platforms exist providing different possibilities
The advent of NGS is reflected by the number of genome projects and data base entries
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
In particular bacterial genome projects boost since 2008
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24
Source httpgenomesonlineorgcgi-binGOLDindexcgi
Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi
Incomplete Genome Projects 27988 Archaeal 457
Bacterial 19494 Eukaryal 6413
Last updated 2014-01-24
Source GOLD = Genomes Online Database at the DOE Joint Genome Institute
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
The first nucleotide sequence of a complete biomolecule was the Alanine tRNA of Yeast
by Robert W Holley et al in 1964 Nobel Prize in Physiology and Medicine 1968
(for 77 nt)
Generation 2 The real breakthrough
bull 1975 -1977 the bdquoSanger Sequencingldquo ndash sequencing by synthesis
bull Nobel Prize Chemistry 1980
bull 1977 the bdquoMaxam and Gilbertldquo Sequencing - sequencing by chemical degradation
bull Nobel Prize Chemistry 1980
Development of the Sanger Sequencing
1975 Sanger and Coulson published the +- method Sanger F Coulson AR A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase J Mol Biol 1975 May 2594(3)441ndash448
1977 Sanger Nicklen and Coulson published the chain terminator method F Sanger S Nicklen and A R Coulson DNA sequencing with chain-terminating inhibitors Proc Natl Acad Sci U S Dec 1977 Av74(12) 5463-5467
The Maxam and Gilbert Method based on base specific chemical degradation of end-
labelled DNA-restriction fragments
In the same year (1977) but 10 months before Sanger published the chain terminator Method Maxam and Gilbert published their DNA sequencing method based on chemical degradation of end-labelled DNA restriction fragments A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Maxam and Gilbert sequencing
bull A very robust method
bull Not sensitive to secondary structures
bull Shows base modification
But
bull Requires work with with
bull strong carcinogens and miliCuries of
radioisotopes
bull Is not automatable
bull Very laborious and requires long
exposition times
A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Sanger sequencing
bull Reading the sequence easier
bull No carcinogenic chemicals involved
bull Exposure times were only a few hours
bull the sequencing reactions could be done by
acute the technician
bull but
the natural DNA-Polymerases are sensitive to
secondary structures and stretches of
homopolymeric nucleotides This changed
only when the bdquosequenasesldquo were invented
Sanger-sequencing has won the race
Maxam and Gilbert
Number of citations
- 7690 times
Sanger Nicklen and Coulson
Number of citations
ndash 62757 times
Source Google Scholar
Generation 3 on line sequencing
- number of different techniques
- all based on fluorescently labelled DNA framents which could be detected and tranferred automatically to a computer
- automated bdquobase callingldquo
bdquoClassicalldquo on line sequencing is still in use
bull The demand is still increasing
bull Results are robust low error rate lt 11000-110000 bp
bull Up to 1500 nt readable in a row
bull Cost per sample ~ 3-5 euro (014 Cent Bpds)
bull Comprehensive service available commercially
Generation 4
Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod
of the yearldquo
bull introduces a new dimension in sequence determination
bull Several platforms exist providing different possibilities
The advent of NGS is reflected by the number of genome projects and data base entries
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
In particular bacterial genome projects boost since 2008
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24
Source httpgenomesonlineorgcgi-binGOLDindexcgi
Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi
Incomplete Genome Projects 27988 Archaeal 457
Bacterial 19494 Eukaryal 6413
Last updated 2014-01-24
Source GOLD = Genomes Online Database at the DOE Joint Genome Institute
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Generation 2 The real breakthrough
bull 1975 -1977 the bdquoSanger Sequencingldquo ndash sequencing by synthesis
bull Nobel Prize Chemistry 1980
bull 1977 the bdquoMaxam and Gilbertldquo Sequencing - sequencing by chemical degradation
bull Nobel Prize Chemistry 1980
Development of the Sanger Sequencing
1975 Sanger and Coulson published the +- method Sanger F Coulson AR A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase J Mol Biol 1975 May 2594(3)441ndash448
1977 Sanger Nicklen and Coulson published the chain terminator method F Sanger S Nicklen and A R Coulson DNA sequencing with chain-terminating inhibitors Proc Natl Acad Sci U S Dec 1977 Av74(12) 5463-5467
The Maxam and Gilbert Method based on base specific chemical degradation of end-
labelled DNA-restriction fragments
In the same year (1977) but 10 months before Sanger published the chain terminator Method Maxam and Gilbert published their DNA sequencing method based on chemical degradation of end-labelled DNA restriction fragments A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Maxam and Gilbert sequencing
bull A very robust method
bull Not sensitive to secondary structures
bull Shows base modification
But
bull Requires work with with
bull strong carcinogens and miliCuries of
radioisotopes
bull Is not automatable
bull Very laborious and requires long
exposition times
A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Sanger sequencing
bull Reading the sequence easier
bull No carcinogenic chemicals involved
bull Exposure times were only a few hours
bull the sequencing reactions could be done by
acute the technician
bull but
the natural DNA-Polymerases are sensitive to
secondary structures and stretches of
homopolymeric nucleotides This changed
only when the bdquosequenasesldquo were invented
Sanger-sequencing has won the race
Maxam and Gilbert
Number of citations
- 7690 times
Sanger Nicklen and Coulson
Number of citations
ndash 62757 times
Source Google Scholar
Generation 3 on line sequencing
- number of different techniques
- all based on fluorescently labelled DNA framents which could be detected and tranferred automatically to a computer
- automated bdquobase callingldquo
bdquoClassicalldquo on line sequencing is still in use
bull The demand is still increasing
bull Results are robust low error rate lt 11000-110000 bp
bull Up to 1500 nt readable in a row
bull Cost per sample ~ 3-5 euro (014 Cent Bpds)
bull Comprehensive service available commercially
Generation 4
Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod
of the yearldquo
bull introduces a new dimension in sequence determination
bull Several platforms exist providing different possibilities
The advent of NGS is reflected by the number of genome projects and data base entries
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
In particular bacterial genome projects boost since 2008
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24
Source httpgenomesonlineorgcgi-binGOLDindexcgi
Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi
Incomplete Genome Projects 27988 Archaeal 457
Bacterial 19494 Eukaryal 6413
Last updated 2014-01-24
Source GOLD = Genomes Online Database at the DOE Joint Genome Institute
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Development of the Sanger Sequencing
1975 Sanger and Coulson published the +- method Sanger F Coulson AR A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase J Mol Biol 1975 May 2594(3)441ndash448
1977 Sanger Nicklen and Coulson published the chain terminator method F Sanger S Nicklen and A R Coulson DNA sequencing with chain-terminating inhibitors Proc Natl Acad Sci U S Dec 1977 Av74(12) 5463-5467
The Maxam and Gilbert Method based on base specific chemical degradation of end-
labelled DNA-restriction fragments
In the same year (1977) but 10 months before Sanger published the chain terminator Method Maxam and Gilbert published their DNA sequencing method based on chemical degradation of end-labelled DNA restriction fragments A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Maxam and Gilbert sequencing
bull A very robust method
bull Not sensitive to secondary structures
bull Shows base modification
But
bull Requires work with with
bull strong carcinogens and miliCuries of
radioisotopes
bull Is not automatable
bull Very laborious and requires long
exposition times
A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Sanger sequencing
bull Reading the sequence easier
bull No carcinogenic chemicals involved
bull Exposure times were only a few hours
bull the sequencing reactions could be done by
acute the technician
bull but
the natural DNA-Polymerases are sensitive to
secondary structures and stretches of
homopolymeric nucleotides This changed
only when the bdquosequenasesldquo were invented
Sanger-sequencing has won the race
Maxam and Gilbert
Number of citations
- 7690 times
Sanger Nicklen and Coulson
Number of citations
ndash 62757 times
Source Google Scholar
Generation 3 on line sequencing
- number of different techniques
- all based on fluorescently labelled DNA framents which could be detected and tranferred automatically to a computer
- automated bdquobase callingldquo
bdquoClassicalldquo on line sequencing is still in use
bull The demand is still increasing
bull Results are robust low error rate lt 11000-110000 bp
bull Up to 1500 nt readable in a row
bull Cost per sample ~ 3-5 euro (014 Cent Bpds)
bull Comprehensive service available commercially
Generation 4
Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod
of the yearldquo
bull introduces a new dimension in sequence determination
bull Several platforms exist providing different possibilities
The advent of NGS is reflected by the number of genome projects and data base entries
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
In particular bacterial genome projects boost since 2008
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24
Source httpgenomesonlineorgcgi-binGOLDindexcgi
Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi
Incomplete Genome Projects 27988 Archaeal 457
Bacterial 19494 Eukaryal 6413
Last updated 2014-01-24
Source GOLD = Genomes Online Database at the DOE Joint Genome Institute
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
The Maxam and Gilbert Method based on base specific chemical degradation of end-
labelled DNA-restriction fragments
In the same year (1977) but 10 months before Sanger published the chain terminator Method Maxam and Gilbert published their DNA sequencing method based on chemical degradation of end-labelled DNA restriction fragments A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Maxam and Gilbert sequencing
bull A very robust method
bull Not sensitive to secondary structures
bull Shows base modification
But
bull Requires work with with
bull strong carcinogens and miliCuries of
radioisotopes
bull Is not automatable
bull Very laborious and requires long
exposition times
A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Sanger sequencing
bull Reading the sequence easier
bull No carcinogenic chemicals involved
bull Exposure times were only a few hours
bull the sequencing reactions could be done by
acute the technician
bull but
the natural DNA-Polymerases are sensitive to
secondary structures and stretches of
homopolymeric nucleotides This changed
only when the bdquosequenasesldquo were invented
Sanger-sequencing has won the race
Maxam and Gilbert
Number of citations
- 7690 times
Sanger Nicklen and Coulson
Number of citations
ndash 62757 times
Source Google Scholar
Generation 3 on line sequencing
- number of different techniques
- all based on fluorescently labelled DNA framents which could be detected and tranferred automatically to a computer
- automated bdquobase callingldquo
bdquoClassicalldquo on line sequencing is still in use
bull The demand is still increasing
bull Results are robust low error rate lt 11000-110000 bp
bull Up to 1500 nt readable in a row
bull Cost per sample ~ 3-5 euro (014 Cent Bpds)
bull Comprehensive service available commercially
Generation 4
Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod
of the yearldquo
bull introduces a new dimension in sequence determination
bull Several platforms exist providing different possibilities
The advent of NGS is reflected by the number of genome projects and data base entries
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
In particular bacterial genome projects boost since 2008
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24
Source httpgenomesonlineorgcgi-binGOLDindexcgi
Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi
Incomplete Genome Projects 27988 Archaeal 457
Bacterial 19494 Eukaryal 6413
Last updated 2014-01-24
Source GOLD = Genomes Online Database at the DOE Joint Genome Institute
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Which sequencing method was superior
Maxam and Gilbert sequencing
bull A very robust method
bull Not sensitive to secondary structures
bull Shows base modification
But
bull Requires work with with
bull strong carcinogens and miliCuries of
radioisotopes
bull Is not automatable
bull Very laborious and requires long
exposition times
A M Maxam W Gilbert A new method for sequencing DNA Proc Natl Acad Sci U S A 1977 February 74(2) 560ndash564
Which sequencing method was superior
Sanger sequencing
bull Reading the sequence easier
bull No carcinogenic chemicals involved
bull Exposure times were only a few hours
bull the sequencing reactions could be done by
acute the technician
bull but
the natural DNA-Polymerases are sensitive to
secondary structures and stretches of
homopolymeric nucleotides This changed
only when the bdquosequenasesldquo were invented
Sanger-sequencing has won the race
Maxam and Gilbert
Number of citations
- 7690 times
Sanger Nicklen and Coulson
Number of citations
ndash 62757 times
Source Google Scholar
Generation 3 on line sequencing
- number of different techniques
- all based on fluorescently labelled DNA framents which could be detected and tranferred automatically to a computer
- automated bdquobase callingldquo
bdquoClassicalldquo on line sequencing is still in use
bull The demand is still increasing
bull Results are robust low error rate lt 11000-110000 bp
bull Up to 1500 nt readable in a row
bull Cost per sample ~ 3-5 euro (014 Cent Bpds)
bull Comprehensive service available commercially
Generation 4
Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod
of the yearldquo
bull introduces a new dimension in sequence determination
bull Several platforms exist providing different possibilities
The advent of NGS is reflected by the number of genome projects and data base entries
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
In particular bacterial genome projects boost since 2008
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24
Source httpgenomesonlineorgcgi-binGOLDindexcgi
Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi
Incomplete Genome Projects 27988 Archaeal 457
Bacterial 19494 Eukaryal 6413
Last updated 2014-01-24
Source GOLD = Genomes Online Database at the DOE Joint Genome Institute
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Which sequencing method was superior
Sanger sequencing
bull Reading the sequence easier
bull No carcinogenic chemicals involved
bull Exposure times were only a few hours
bull the sequencing reactions could be done by
acute the technician
bull but
the natural DNA-Polymerases are sensitive to
secondary structures and stretches of
homopolymeric nucleotides This changed
only when the bdquosequenasesldquo were invented
Sanger-sequencing has won the race
Maxam and Gilbert
Number of citations
- 7690 times
Sanger Nicklen and Coulson
Number of citations
ndash 62757 times
Source Google Scholar
Generation 3 on line sequencing
- number of different techniques
- all based on fluorescently labelled DNA framents which could be detected and tranferred automatically to a computer
- automated bdquobase callingldquo
bdquoClassicalldquo on line sequencing is still in use
bull The demand is still increasing
bull Results are robust low error rate lt 11000-110000 bp
bull Up to 1500 nt readable in a row
bull Cost per sample ~ 3-5 euro (014 Cent Bpds)
bull Comprehensive service available commercially
Generation 4
Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod
of the yearldquo
bull introduces a new dimension in sequence determination
bull Several platforms exist providing different possibilities
The advent of NGS is reflected by the number of genome projects and data base entries
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
In particular bacterial genome projects boost since 2008
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24
Source httpgenomesonlineorgcgi-binGOLDindexcgi
Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi
Incomplete Genome Projects 27988 Archaeal 457
Bacterial 19494 Eukaryal 6413
Last updated 2014-01-24
Source GOLD = Genomes Online Database at the DOE Joint Genome Institute
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Sanger-sequencing has won the race
Maxam and Gilbert
Number of citations
- 7690 times
Sanger Nicklen and Coulson
Number of citations
ndash 62757 times
Source Google Scholar
Generation 3 on line sequencing
- number of different techniques
- all based on fluorescently labelled DNA framents which could be detected and tranferred automatically to a computer
- automated bdquobase callingldquo
bdquoClassicalldquo on line sequencing is still in use
bull The demand is still increasing
bull Results are robust low error rate lt 11000-110000 bp
bull Up to 1500 nt readable in a row
bull Cost per sample ~ 3-5 euro (014 Cent Bpds)
bull Comprehensive service available commercially
Generation 4
Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod
of the yearldquo
bull introduces a new dimension in sequence determination
bull Several platforms exist providing different possibilities
The advent of NGS is reflected by the number of genome projects and data base entries
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
In particular bacterial genome projects boost since 2008
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24
Source httpgenomesonlineorgcgi-binGOLDindexcgi
Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi
Incomplete Genome Projects 27988 Archaeal 457
Bacterial 19494 Eukaryal 6413
Last updated 2014-01-24
Source GOLD = Genomes Online Database at the DOE Joint Genome Institute
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Generation 3 on line sequencing
- number of different techniques
- all based on fluorescently labelled DNA framents which could be detected and tranferred automatically to a computer
- automated bdquobase callingldquo
bdquoClassicalldquo on line sequencing is still in use
bull The demand is still increasing
bull Results are robust low error rate lt 11000-110000 bp
bull Up to 1500 nt readable in a row
bull Cost per sample ~ 3-5 euro (014 Cent Bpds)
bull Comprehensive service available commercially
Generation 4
Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod
of the yearldquo
bull introduces a new dimension in sequence determination
bull Several platforms exist providing different possibilities
The advent of NGS is reflected by the number of genome projects and data base entries
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
In particular bacterial genome projects boost since 2008
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24
Source httpgenomesonlineorgcgi-binGOLDindexcgi
Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi
Incomplete Genome Projects 27988 Archaeal 457
Bacterial 19494 Eukaryal 6413
Last updated 2014-01-24
Source GOLD = Genomes Online Database at the DOE Joint Genome Institute
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
bdquoClassicalldquo on line sequencing is still in use
bull The demand is still increasing
bull Results are robust low error rate lt 11000-110000 bp
bull Up to 1500 nt readable in a row
bull Cost per sample ~ 3-5 euro (014 Cent Bpds)
bull Comprehensive service available commercially
Generation 4
Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod
of the yearldquo
bull introduces a new dimension in sequence determination
bull Several platforms exist providing different possibilities
The advent of NGS is reflected by the number of genome projects and data base entries
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
In particular bacterial genome projects boost since 2008
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24
Source httpgenomesonlineorgcgi-binGOLDindexcgi
Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi
Incomplete Genome Projects 27988 Archaeal 457
Bacterial 19494 Eukaryal 6413
Last updated 2014-01-24
Source GOLD = Genomes Online Database at the DOE Joint Genome Institute
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Generation 4
Next generation sequencing (NGS) bull 2007 NGS selected by Nature as the bdquomethod
of the yearldquo
bull introduces a new dimension in sequence determination
bull Several platforms exist providing different possibilities
The advent of NGS is reflected by the number of genome projects and data base entries
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
In particular bacterial genome projects boost since 2008
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24
Source httpgenomesonlineorgcgi-binGOLDindexcgi
Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi
Incomplete Genome Projects 27988 Archaeal 457
Bacterial 19494 Eukaryal 6413
Last updated 2014-01-24
Source GOLD = Genomes Online Database at the DOE Joint Genome Institute
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
The advent of NGS is reflected by the number of genome projects and data base entries
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
In particular bacterial genome projects boost since 2008
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24
Source httpgenomesonlineorgcgi-binGOLDindexcgi
Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi
Incomplete Genome Projects 27988 Archaeal 457
Bacterial 19494 Eukaryal 6413
Last updated 2014-01-24
Source GOLD = Genomes Online Database at the DOE Joint Genome Institute
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
In particular bacterial genome projects boost since 2008
httpwwwgenomesonlineorgcgi-binGOLDindexcgipage_requested=Statistics
Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24
Source httpgenomesonlineorgcgi-binGOLDindexcgi
Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi
Incomplete Genome Projects 27988 Archaeal 457
Bacterial 19494 Eukaryal 6413
Last updated 2014-01-24
Source GOLD = Genomes Online Database at the DOE Joint Genome Institute
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Complete Genome Projects 12725 Archaeal 317 Bacterial 12096 Eukaryal 312 Finished 2876 Permanent Draft 9849 Last updated 2014-01-24
Source httpgenomesonlineorgcgi-binGOLDindexcgi
Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi
Incomplete Genome Projects 27988 Archaeal 457
Bacterial 19494 Eukaryal 6413
Last updated 2014-01-24
Source GOLD = Genomes Online Database at the DOE Joint Genome Institute
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Genome Projects httpwwwgenomesonlineorgcgi-binGOLDindexcgi
Incomplete Genome Projects 27988 Archaeal 457
Bacterial 19494 Eukaryal 6413
Last updated 2014-01-24
Source GOLD = Genomes Online Database at the DOE Joint Genome Institute
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
NGS has revolutionized genome science
bull Reduction of costs
bull Reduction of time
bull Reduction of labour
bull Increase in bioinformatical challenge
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
The different platforms
The genome scale
bull 454Roche GenomeSequencer FLX
bull ABI SOLiD Sequencing System
bull IlluminaSolexa Hi-Seq20002500
bull Ion Torrent Proton
bull Pacific Bioscience
bull (Helicos)
The bench top scale
bull 454 GS JuniorRoche
bull Illumina MiSeq
bull Illumina NextSeq500
bull Ion Torrent PGMLife Technologies
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
454Roche GS FLX
The basis is Emulsion PCR and Pyrosequencing
sst-DNA single-stranded template DNA
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
The number of sequences is depending on the number of wells in plate
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
454Roche GS FLX
Pyrosequencing
AcircPS = Adenosinephosphosulfate
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Pyrosequencing is not suitable for sequencing oligopolymers ngt6-7
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
GS FLX+ System
Sequencing Kit New GS FLX Titanium XL+
GS FLX Titanium XLR70
Read Length Up to 1000 bp Up to 600 bp
Mode Read Length 700 bp 450 bp
Throughput Profile
- 85 of total bases from reads gt500 bp - 45 of total bases from reads gt700 bp
- 85 of total bases from reads gt 300 bp - 20 of total bases from reads gt 500 bp
Typical Throughput 700 Mb 450 Mb
Reads per Run ~1000000 shotgun ~1000000 shotgun ~700000 amplicon
Consensus Accuracy 99997 99995
Run Time 23 hours 10 hours
Sample Input gDNA or cDNA
gDNA cDNA or amplicons (PCR products)
Multiplexing Multiplex Identifiers (MIDs) 132 Gaskets 2 4 8 16 regions
Data from Roche http454comproductsgs-flx-system
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
454Roche GS FLX Titanium
bull Advantages
bull Long read length gt400 nt up to 1000
bull Low error rate but sensitive to homooligomers
bull Disadvantages
bull Data output lt 07 Gb
bull Cost per Gigabase is highest among all systems
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Applied Biosystems SOLiDTM-Sequencing bull SOLiD = Sequencing by Oligonucleotide Ligation and Detection
Template preparation Emulsion PCR Sequencing Hybridization and ligation
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
By successive rounds labelled oligonucleotide ligation to the template each base in the
template is determined twice
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Process of SOLiD Sequencing
Figure from Clinical Chemistry April 2009 vol 55 no 4 641-658
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Each base is sequenced twice
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Applied Biosystems SOLiDTM Sequencing
bull Advantage bull Very good data quality since every base
sequenced twice (9999 correct) bull High data output ~ Solid4TMhq 300 Gbrun 14d bull High number of possible multiplexing (up to
1536 sample per run) bull Cost effective 2000 eurohuman genome
bull Disadvantage bull Maximum read length is 75 bases bull 14 days run time for 2x75 bases
Data from httpwww3appliedbiosystemscomcmsgroupsmcb_marketingdocumentsgeneraldocumentscms_061241pdf
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
IlluminaSolexaTM-Sequencing
bull Sequencing by Synthesis
bull Modified chain terminating method
bull Bridge amplification
bull Paired end and mate pair libraries possible
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
IlluminaSolexaTM-Sequencing Clustering and sequencing
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
IlluminaSolexaTM-Sequencing
Advantages (Hi-SeqTM 20002500)
Very high data output gt 400 Mio reads PElane ~ 600 Gigabaserun
Read length PE 2x150 bases (increasing)
Cost per Gb ~ lt50euro or 1500eurohuman genome
Disadvantages
bull Hardware investment is high (~600000 euro plus periphery)
bull Medium high error rate (~05 increasing with read length)
bull High maintenance costs (service contract gt80000 euroyear)
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
bull Based on sequencing by Synthesis
bull Available since 2010
bull Emulsion PCR for library construction
bull Beads with amplified molecules are primed with an adapter
bull Beads are put in an bdquoIon Chipldquo that is sensitive for H+-Ions
bull Incorporation of a nucleotide produces an H+-Ion which is measured by the chip
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Annual Reviews
G A T C
Figure modified by E R Schmidt
Ion Torrent NGS by pH-Change Measurement on a Semiconductor Chip
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Life TechnologiesIon Torrent-Sequencing by pH Monitoring
Advantages
Very cost efficient (human genome lt 1000 euro)
Read length 200 bases (increasing)
Very short running times
(~ 2-4 hrs)
Hardware investment is bdquolowldquo (~ 80000 US $)
Disadvantages
bull High error rate (gt10 increasing with read length)
bull Especially sensitive to oligopolymer stretches leading to a high rate of bdquodeletionsldquo
bull Data output medium (depending on chip eg Proton PII = 32 Gb)
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Pacific BiosciencesSingle molecule real time (SMRT)-sequencing
bull Based on sequencing by synthesis on single molecules
bull Available since 2010 bull Special library construction leading to circular
molecules (enables multiple sequencing of the same molecule)
bull Binding of bdquoengineeredldquo DNA-Polymerase in bdquozero-mode waveguideldquo manufactured on a silicon wafer (SMRTTM-cell)
bull fluorescence labelled dNTP are measured in real time during incorporation
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Zero-mode waveguide
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Pacific BiosciencesSingle molecule polymerase active site monitoring
Advantages
Read length up to 10000 bases (average gt 1000 b)
Very short running times
(~ 2hrs)
Low running cost acc to the company a genome human equivalent bdquoa few hundred dollarsldquo
Disadvantages
bull High error rate (gt10-15 for single pass sequencing repeated sequencing lowers error rate to 2-3)
bull Significant investment in hardware (gt600 keuro)
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
HelicosTM-Sequencing (16 November 2012 bancruptcy protection chapter 11)
bull Sequencing by Synthesis with single molecules as templates
bull Modified chain terminating method
bull Bridge amplification
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Benchtop NGS Sequencing
bull Illumina Mi-SeqTM
bull Roche 454 JuniorTM
bull Ion Torrent PGMTM
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Platform List price Approximate cost per
run
Minimum throughput (read length)
Run time CostMb Mbh
454 GS Junior $108000 $1100 35 Mb (400 bases)
8 h $31 44
Ion Torrent PGM (314 chip)
$80490ab $225c 10 Mb (100 bases)
3 h $225 33
(316 chip) $425 100 Mbd (100 bases)
3 h $425 333
(318 chip) $625 1000 Mb (100 bases)
3 h $063 3333
MiSeq $125000 $750 1500 Mb (2 times 150 bases)
27 h $05 555
Costs and Performance of benchtop NGS Table 1 Price comparison of benchtop instruments and sequencing runs
From Performance comparison of benchtop high-throughput sequencing platform Nicholas J Loman Raju V Misra Timothy J Dallman Chrystala Constantinidou Saheer Gharbia John Wain amp Mark J Pallen Nature Biotechnology30434ndash439 (2012) doi101038nbt2198
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Updating benchtop sequencing performance comparison Sebastian Juumlnemann Fritz Joachim Sedlazeck Karola Prior Andreas Albersmeier Uwe John Joumlrn Kalinowski Alexander Mellmann Alexander Goesmann Arndt von Haeseler Jens Stoye amp Dag Harmsen Nature Biotechnology 31 294ndash296 (2013) doi101038nbt2522 Published online 05 April 2013
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
httpwwwgenomegovimagescontentcost_per_megabasejpg
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Generation 5 Future Technology Nanopore Sequencing
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
bull A method of controlled translocation of the strand through the nanopore is needed Oxford Nanopore uses proprietary highly processive enzymes to ratchet DNA through the nanopore Watch our movie for more information Oxford Nanopore has not disclosed the proprietary nanopore and enzyme machinery used in its GridION and MinION system Oxford Nanopore has not signed a commercialisation agreement for strand sequencing and intends to commercialise strand sequencing products independently
When a DNA polymer passes through a nanopore a number of individual DNA bases occupy the aperture of the nanopore at any time A successful method of DNA sequencing must identify the sequence of individual bases within this strand Oxford Nanopore has engineered bespoke nanopores and data analysis algorithms are used to translate the characteristic electronic signals into DNA sequence data
In Oxford Nanopores strand sequencing method a single-stranded DNA polymer is passed through a protein nanopore and individual DNA bases on the strand are identified in sequence as the DNA molecule passes through
httpswwwnanoporetechcomhome From
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Applications of NGS
bull Genome Resequencing and SNP Detection bull Genome De novo sequencing bull Transcriptome sequencing bull ChIP-sequencing Histonmethylation bull Bisulfate-sequencing for methylation analysis bull Exome enrichment sequencing bull Small RNA sequencing bull Genotyping by Sequencing (GBS RAD) reduced
complexity sequencing bull Ribosome profiling
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
No summary table
Equipment and technologies are too diverse so a good advice would be Discuss your project with people having experience with one or the other platform NGS is a fantastic novel technology which provides completely new possibilities Projects that have been even unthinkable a few years ago are now easy going
Thank you very much for your attention
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
Benjamin Rieger bioinformatician
Nicole Naumann technical assistant
Rudolf Baader technical assistant
Steffen Rapp NGS Unit manager
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