Embed Size (px)
Citation preview
By Muhammad Qayash KhanPhD Zoology Ist Semester
13-arid-3853
Next-Generation Sequencing Methodology and Application
DNA Sequencing
The process of determining the order of the nucleotide bases along a DNA strand is called sequencingIn 1977 twenty-four years after the discovery of the structure of DNA two separate methods for sequencing DNA were developed the chain termination method and the chemical degradation method
The chain termination method is the method more commonly used
Breakthroughs in Technology
bull An essential tool in the molecular biology toolkit is the ability to read the base sequence of DNA molecules
bull Rapid DNA sequencing in the 1970sndash Sangerndash Gilbert and Maxam
bull Microarrays in late 1990s and 2000sndash cDNA arraysndash oligonucleotide arrays
Dideoxy (Sanger) Method
bull 4 Steps1 Denaturation 2 Primer attachment and extension of bases3 Termination4 Gel electrophoresis
Chain Termination Method
This method is based on the principle that single-stranded DNA molecules that differ in length by just a single nucleotide can be separated from one another using polyacrylamide gel electrophoresis (CE)bull A DNA templatebull A primer tagged with a mildly radioactive molecule or a light-emitting chemicalbull DNA polymerase--an enzyme that drives the synthesis of DNAbull Four deoxynucleotides (G A C T)bull One dideoxynucleotide either ddG ddA ddC or ddT
Overview Dideoxy (Sanger) Method
1
4
32
Gel electrophoresis
5
Dideoxy Method
bull Run four separate reactions each with different ddNTPsbull Run on a gel in four separate lanesbull Read the gel from the bottom up
Sanger sequencing vs NGS
bull Sanger sequencing is limiting because of the high cost and labor intensity
bull The development of next-generation sequencing (NGS) technologies has facilitated the collection of large amounts of nucleotide information in sequence read-length from 30 to 1500 nucleotides (nt) for hundreds of thousands to millions of DNA molecules simultaneously
bull In parallel the bioinformatics tools required to analyze these large datasets and identify unique gene sequences have also significantly improved
What is Next-Generation Sequencing
bull The advances of sequencing technologies have successfully contributed in elucidating the function of the human genome
bull NGS technologies have gained the capacity to sequence giga bases of DNA in a high-throughput and highly efficient manner that has not been possible using traditional Sanger sequencing
bull While Sanger is based on gel separation of chain-terminated fragments from enzymatic synthesis most NGS techniques are based on locally bound nano-clusters of template DNA and incorporation of fluorescent-labeled nucleotides by DNA polymerases or ligation processes
Microarrays
bull Extremely successfulbull Popular applications Gene expression profiling DNA
copy number (comparative genomic hybridization) SNPs microRNAs ChIP-chip (tiling arrays) splicing (exon arrays)
bull Disadvantagesbull One must know the sequences to design the arraybull Even if one knows the sequences one cannot fit all of
them in a small number of arraysbull High noise level due to cross-hybrization non-linearity
etc
Microarrays
bull cDNA arrays bull oligonucleotide arrays
PLATFORMS AND TECHNOLOGY
bull There are five leading instruments that can be classified as part of the NGS technologies
bull the 454 GS FLX bull the Ion Torrentbull the SOLiD bull the Illumina bull and the more recently released PacBio
instrument
NGS platform properties
bull NGS platforms can be distinguished from each other based on
bull the chemistry employed for sequencing bull the amount of sequence information
producedbull the length of each sequence read bull the overall price per nt
454 GENOME SEQUENCER-FLXTM
bull The 454 pyro sequencer manufactured by Rochebull Principlebull Pyro sequencing bull a method of detecting single nucleotide addition
by capturing the emission of light produced from the release of the by-product pyrophosphate during the polymerization of the DNA molecule (Droege and Hill 2008 Rothberg and Leamon 2008)
Pyrosequencing Solid Phase method
1st MethodSolid Phase
Immobilized DNA 3 enzymes Wash step to remove nucleotides after each
addition
Pyrosequencing liquid phase method
2nd MethodLiquid Phase
3 enzymes + apyrase (nucleotide degradation enzyme)Eliminates need for washing step
bull In the well of a microtiter platebull primed DNA templatebull 4 enzymesbull Nucleotides are added stepwisebull Nucleotide-degrading enzyme degrade previous nucleotides
Pyrosequencing Method
Pyrosequencing Results
Example of NGS-BASED STUDIES
ION TORRENT SEMICONDUCTOR SEQUENCER
bull Modified version of the 454 pyrosequencing approach and operates
bull Based on the same sequencing chemistry except that it makes use of the H+ that is released with every nucleotide incorporated instead of the pyrophosphate (Rothberg et al2011)
bull Significantly reduces the cost of sequencing since luciferase and other costly enzymes and scanners are not needed
bull To date this instrument can sequence about 100nt but It should soon be able to read sequence lengths up to 200nt (IonTorrentSystemInc2)
SOLiD TM SYSTEM
bull The SOLiD genome sequencer from Applied Biosystems uses an emPCR process similar to 454 but parallel DNA sequencing is achieved by repeatedly ligating two-nucleotide probes instead of a sequencing reaction catalyzed by DNA polymerase (Morozova and Marra 2008)
bull The two-nucleotide probes are used to query adjacent bases on the DNA fragment therefore each nucleotide is actually probed twice
bull This system is designed to make sequence calls on two signals per base rather than one resulting in a lower error rate
bull Read approximately 35 nucleotides (Morozova and Marra 2008) but current versions of the instrument have increased the read-length to about 50 nucleotides (Applied Biosystems)
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
DNA Sequencing
The process of determining the order of the nucleotide bases along a DNA strand is called sequencingIn 1977 twenty-four years after the discovery of the structure of DNA two separate methods for sequencing DNA were developed the chain termination method and the chemical degradation method
The chain termination method is the method more commonly used
Breakthroughs in Technology
bull An essential tool in the molecular biology toolkit is the ability to read the base sequence of DNA molecules
bull Rapid DNA sequencing in the 1970sndash Sangerndash Gilbert and Maxam
bull Microarrays in late 1990s and 2000sndash cDNA arraysndash oligonucleotide arrays
Dideoxy (Sanger) Method
bull 4 Steps1 Denaturation 2 Primer attachment and extension of bases3 Termination4 Gel electrophoresis
Chain Termination Method
This method is based on the principle that single-stranded DNA molecules that differ in length by just a single nucleotide can be separated from one another using polyacrylamide gel electrophoresis (CE)bull A DNA templatebull A primer tagged with a mildly radioactive molecule or a light-emitting chemicalbull DNA polymerase--an enzyme that drives the synthesis of DNAbull Four deoxynucleotides (G A C T)bull One dideoxynucleotide either ddG ddA ddC or ddT
Overview Dideoxy (Sanger) Method
1
4
32
Gel electrophoresis
5
Dideoxy Method
bull Run four separate reactions each with different ddNTPsbull Run on a gel in four separate lanesbull Read the gel from the bottom up
Sanger sequencing vs NGS
bull Sanger sequencing is limiting because of the high cost and labor intensity
bull The development of next-generation sequencing (NGS) technologies has facilitated the collection of large amounts of nucleotide information in sequence read-length from 30 to 1500 nucleotides (nt) for hundreds of thousands to millions of DNA molecules simultaneously
bull In parallel the bioinformatics tools required to analyze these large datasets and identify unique gene sequences have also significantly improved
What is Next-Generation Sequencing
bull The advances of sequencing technologies have successfully contributed in elucidating the function of the human genome
bull NGS technologies have gained the capacity to sequence giga bases of DNA in a high-throughput and highly efficient manner that has not been possible using traditional Sanger sequencing
bull While Sanger is based on gel separation of chain-terminated fragments from enzymatic synthesis most NGS techniques are based on locally bound nano-clusters of template DNA and incorporation of fluorescent-labeled nucleotides by DNA polymerases or ligation processes
Microarrays
bull Extremely successfulbull Popular applications Gene expression profiling DNA
copy number (comparative genomic hybridization) SNPs microRNAs ChIP-chip (tiling arrays) splicing (exon arrays)
bull Disadvantagesbull One must know the sequences to design the arraybull Even if one knows the sequences one cannot fit all of
them in a small number of arraysbull High noise level due to cross-hybrization non-linearity
etc
Microarrays
bull cDNA arrays bull oligonucleotide arrays
PLATFORMS AND TECHNOLOGY
bull There are five leading instruments that can be classified as part of the NGS technologies
bull the 454 GS FLX bull the Ion Torrentbull the SOLiD bull the Illumina bull and the more recently released PacBio
instrument
NGS platform properties
bull NGS platforms can be distinguished from each other based on
bull the chemistry employed for sequencing bull the amount of sequence information
producedbull the length of each sequence read bull the overall price per nt
454 GENOME SEQUENCER-FLXTM
bull The 454 pyro sequencer manufactured by Rochebull Principlebull Pyro sequencing bull a method of detecting single nucleotide addition
by capturing the emission of light produced from the release of the by-product pyrophosphate during the polymerization of the DNA molecule (Droege and Hill 2008 Rothberg and Leamon 2008)
Pyrosequencing Solid Phase method
1st MethodSolid Phase
Immobilized DNA 3 enzymes Wash step to remove nucleotides after each
addition
Pyrosequencing liquid phase method
2nd MethodLiquid Phase
3 enzymes + apyrase (nucleotide degradation enzyme)Eliminates need for washing step
bull In the well of a microtiter platebull primed DNA templatebull 4 enzymesbull Nucleotides are added stepwisebull Nucleotide-degrading enzyme degrade previous nucleotides
Pyrosequencing Method
Pyrosequencing Results
Example of NGS-BASED STUDIES
ION TORRENT SEMICONDUCTOR SEQUENCER
bull Modified version of the 454 pyrosequencing approach and operates
bull Based on the same sequencing chemistry except that it makes use of the H+ that is released with every nucleotide incorporated instead of the pyrophosphate (Rothberg et al2011)
bull Significantly reduces the cost of sequencing since luciferase and other costly enzymes and scanners are not needed
bull To date this instrument can sequence about 100nt but It should soon be able to read sequence lengths up to 200nt (IonTorrentSystemInc2)
SOLiD TM SYSTEM
bull The SOLiD genome sequencer from Applied Biosystems uses an emPCR process similar to 454 but parallel DNA sequencing is achieved by repeatedly ligating two-nucleotide probes instead of a sequencing reaction catalyzed by DNA polymerase (Morozova and Marra 2008)
bull The two-nucleotide probes are used to query adjacent bases on the DNA fragment therefore each nucleotide is actually probed twice
bull This system is designed to make sequence calls on two signals per base rather than one resulting in a lower error rate
bull Read approximately 35 nucleotides (Morozova and Marra 2008) but current versions of the instrument have increased the read-length to about 50 nucleotides (Applied Biosystems)
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
Breakthroughs in Technology
bull An essential tool in the molecular biology toolkit is the ability to read the base sequence of DNA molecules
bull Rapid DNA sequencing in the 1970sndash Sangerndash Gilbert and Maxam
bull Microarrays in late 1990s and 2000sndash cDNA arraysndash oligonucleotide arrays
Dideoxy (Sanger) Method
bull 4 Steps1 Denaturation 2 Primer attachment and extension of bases3 Termination4 Gel electrophoresis
Chain Termination Method
This method is based on the principle that single-stranded DNA molecules that differ in length by just a single nucleotide can be separated from one another using polyacrylamide gel electrophoresis (CE)bull A DNA templatebull A primer tagged with a mildly radioactive molecule or a light-emitting chemicalbull DNA polymerase--an enzyme that drives the synthesis of DNAbull Four deoxynucleotides (G A C T)bull One dideoxynucleotide either ddG ddA ddC or ddT
Overview Dideoxy (Sanger) Method
1
4
32
Gel electrophoresis
5
Dideoxy Method
bull Run four separate reactions each with different ddNTPsbull Run on a gel in four separate lanesbull Read the gel from the bottom up
Sanger sequencing vs NGS
bull Sanger sequencing is limiting because of the high cost and labor intensity
bull The development of next-generation sequencing (NGS) technologies has facilitated the collection of large amounts of nucleotide information in sequence read-length from 30 to 1500 nucleotides (nt) for hundreds of thousands to millions of DNA molecules simultaneously
bull In parallel the bioinformatics tools required to analyze these large datasets and identify unique gene sequences have also significantly improved
What is Next-Generation Sequencing
bull The advances of sequencing technologies have successfully contributed in elucidating the function of the human genome
bull NGS technologies have gained the capacity to sequence giga bases of DNA in a high-throughput and highly efficient manner that has not been possible using traditional Sanger sequencing
bull While Sanger is based on gel separation of chain-terminated fragments from enzymatic synthesis most NGS techniques are based on locally bound nano-clusters of template DNA and incorporation of fluorescent-labeled nucleotides by DNA polymerases or ligation processes
Microarrays
bull Extremely successfulbull Popular applications Gene expression profiling DNA
copy number (comparative genomic hybridization) SNPs microRNAs ChIP-chip (tiling arrays) splicing (exon arrays)
bull Disadvantagesbull One must know the sequences to design the arraybull Even if one knows the sequences one cannot fit all of
them in a small number of arraysbull High noise level due to cross-hybrization non-linearity
etc
Microarrays
bull cDNA arrays bull oligonucleotide arrays
PLATFORMS AND TECHNOLOGY
bull There are five leading instruments that can be classified as part of the NGS technologies
bull the 454 GS FLX bull the Ion Torrentbull the SOLiD bull the Illumina bull and the more recently released PacBio
instrument
NGS platform properties
bull NGS platforms can be distinguished from each other based on
bull the chemistry employed for sequencing bull the amount of sequence information
producedbull the length of each sequence read bull the overall price per nt
454 GENOME SEQUENCER-FLXTM
bull The 454 pyro sequencer manufactured by Rochebull Principlebull Pyro sequencing bull a method of detecting single nucleotide addition
by capturing the emission of light produced from the release of the by-product pyrophosphate during the polymerization of the DNA molecule (Droege and Hill 2008 Rothberg and Leamon 2008)
Pyrosequencing Solid Phase method
1st MethodSolid Phase
Immobilized DNA 3 enzymes Wash step to remove nucleotides after each
addition
Pyrosequencing liquid phase method
2nd MethodLiquid Phase
3 enzymes + apyrase (nucleotide degradation enzyme)Eliminates need for washing step
bull In the well of a microtiter platebull primed DNA templatebull 4 enzymesbull Nucleotides are added stepwisebull Nucleotide-degrading enzyme degrade previous nucleotides
Pyrosequencing Method
Pyrosequencing Results
Example of NGS-BASED STUDIES
ION TORRENT SEMICONDUCTOR SEQUENCER
bull Modified version of the 454 pyrosequencing approach and operates
bull Based on the same sequencing chemistry except that it makes use of the H+ that is released with every nucleotide incorporated instead of the pyrophosphate (Rothberg et al2011)
bull Significantly reduces the cost of sequencing since luciferase and other costly enzymes and scanners are not needed
bull To date this instrument can sequence about 100nt but It should soon be able to read sequence lengths up to 200nt (IonTorrentSystemInc2)
SOLiD TM SYSTEM
bull The SOLiD genome sequencer from Applied Biosystems uses an emPCR process similar to 454 but parallel DNA sequencing is achieved by repeatedly ligating two-nucleotide probes instead of a sequencing reaction catalyzed by DNA polymerase (Morozova and Marra 2008)
bull The two-nucleotide probes are used to query adjacent bases on the DNA fragment therefore each nucleotide is actually probed twice
bull This system is designed to make sequence calls on two signals per base rather than one resulting in a lower error rate
bull Read approximately 35 nucleotides (Morozova and Marra 2008) but current versions of the instrument have increased the read-length to about 50 nucleotides (Applied Biosystems)
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
Dideoxy (Sanger) Method
bull 4 Steps1 Denaturation 2 Primer attachment and extension of bases3 Termination4 Gel electrophoresis
Chain Termination Method
This method is based on the principle that single-stranded DNA molecules that differ in length by just a single nucleotide can be separated from one another using polyacrylamide gel electrophoresis (CE)bull A DNA templatebull A primer tagged with a mildly radioactive molecule or a light-emitting chemicalbull DNA polymerase--an enzyme that drives the synthesis of DNAbull Four deoxynucleotides (G A C T)bull One dideoxynucleotide either ddG ddA ddC or ddT
Overview Dideoxy (Sanger) Method
1
4
32
Gel electrophoresis
5
Dideoxy Method
bull Run four separate reactions each with different ddNTPsbull Run on a gel in four separate lanesbull Read the gel from the bottom up
Sanger sequencing vs NGS
bull Sanger sequencing is limiting because of the high cost and labor intensity
bull The development of next-generation sequencing (NGS) technologies has facilitated the collection of large amounts of nucleotide information in sequence read-length from 30 to 1500 nucleotides (nt) for hundreds of thousands to millions of DNA molecules simultaneously
bull In parallel the bioinformatics tools required to analyze these large datasets and identify unique gene sequences have also significantly improved
What is Next-Generation Sequencing
bull The advances of sequencing technologies have successfully contributed in elucidating the function of the human genome
bull NGS technologies have gained the capacity to sequence giga bases of DNA in a high-throughput and highly efficient manner that has not been possible using traditional Sanger sequencing
bull While Sanger is based on gel separation of chain-terminated fragments from enzymatic synthesis most NGS techniques are based on locally bound nano-clusters of template DNA and incorporation of fluorescent-labeled nucleotides by DNA polymerases or ligation processes
Microarrays
bull Extremely successfulbull Popular applications Gene expression profiling DNA
copy number (comparative genomic hybridization) SNPs microRNAs ChIP-chip (tiling arrays) splicing (exon arrays)
bull Disadvantagesbull One must know the sequences to design the arraybull Even if one knows the sequences one cannot fit all of
them in a small number of arraysbull High noise level due to cross-hybrization non-linearity
etc
Microarrays
bull cDNA arrays bull oligonucleotide arrays
PLATFORMS AND TECHNOLOGY
bull There are five leading instruments that can be classified as part of the NGS technologies
bull the 454 GS FLX bull the Ion Torrentbull the SOLiD bull the Illumina bull and the more recently released PacBio
instrument
NGS platform properties
bull NGS platforms can be distinguished from each other based on
bull the chemistry employed for sequencing bull the amount of sequence information
producedbull the length of each sequence read bull the overall price per nt
454 GENOME SEQUENCER-FLXTM
bull The 454 pyro sequencer manufactured by Rochebull Principlebull Pyro sequencing bull a method of detecting single nucleotide addition
by capturing the emission of light produced from the release of the by-product pyrophosphate during the polymerization of the DNA molecule (Droege and Hill 2008 Rothberg and Leamon 2008)
Pyrosequencing Solid Phase method
1st MethodSolid Phase
Immobilized DNA 3 enzymes Wash step to remove nucleotides after each
addition
Pyrosequencing liquid phase method
2nd MethodLiquid Phase
3 enzymes + apyrase (nucleotide degradation enzyme)Eliminates need for washing step
bull In the well of a microtiter platebull primed DNA templatebull 4 enzymesbull Nucleotides are added stepwisebull Nucleotide-degrading enzyme degrade previous nucleotides
Pyrosequencing Method
Pyrosequencing Results
Example of NGS-BASED STUDIES
ION TORRENT SEMICONDUCTOR SEQUENCER
bull Modified version of the 454 pyrosequencing approach and operates
bull Based on the same sequencing chemistry except that it makes use of the H+ that is released with every nucleotide incorporated instead of the pyrophosphate (Rothberg et al2011)
bull Significantly reduces the cost of sequencing since luciferase and other costly enzymes and scanners are not needed
bull To date this instrument can sequence about 100nt but It should soon be able to read sequence lengths up to 200nt (IonTorrentSystemInc2)
SOLiD TM SYSTEM
bull The SOLiD genome sequencer from Applied Biosystems uses an emPCR process similar to 454 but parallel DNA sequencing is achieved by repeatedly ligating two-nucleotide probes instead of a sequencing reaction catalyzed by DNA polymerase (Morozova and Marra 2008)
bull The two-nucleotide probes are used to query adjacent bases on the DNA fragment therefore each nucleotide is actually probed twice
bull This system is designed to make sequence calls on two signals per base rather than one resulting in a lower error rate
bull Read approximately 35 nucleotides (Morozova and Marra 2008) but current versions of the instrument have increased the read-length to about 50 nucleotides (Applied Biosystems)
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
Chain Termination Method
This method is based on the principle that single-stranded DNA molecules that differ in length by just a single nucleotide can be separated from one another using polyacrylamide gel electrophoresis (CE)bull A DNA templatebull A primer tagged with a mildly radioactive molecule or a light-emitting chemicalbull DNA polymerase--an enzyme that drives the synthesis of DNAbull Four deoxynucleotides (G A C T)bull One dideoxynucleotide either ddG ddA ddC or ddT
Overview Dideoxy (Sanger) Method
1
4
32
Gel electrophoresis
5
Dideoxy Method
bull Run four separate reactions each with different ddNTPsbull Run on a gel in four separate lanesbull Read the gel from the bottom up
Sanger sequencing vs NGS
bull Sanger sequencing is limiting because of the high cost and labor intensity
bull The development of next-generation sequencing (NGS) technologies has facilitated the collection of large amounts of nucleotide information in sequence read-length from 30 to 1500 nucleotides (nt) for hundreds of thousands to millions of DNA molecules simultaneously
bull In parallel the bioinformatics tools required to analyze these large datasets and identify unique gene sequences have also significantly improved
What is Next-Generation Sequencing
bull The advances of sequencing technologies have successfully contributed in elucidating the function of the human genome
bull NGS technologies have gained the capacity to sequence giga bases of DNA in a high-throughput and highly efficient manner that has not been possible using traditional Sanger sequencing
bull While Sanger is based on gel separation of chain-terminated fragments from enzymatic synthesis most NGS techniques are based on locally bound nano-clusters of template DNA and incorporation of fluorescent-labeled nucleotides by DNA polymerases or ligation processes
Microarrays
bull Extremely successfulbull Popular applications Gene expression profiling DNA
copy number (comparative genomic hybridization) SNPs microRNAs ChIP-chip (tiling arrays) splicing (exon arrays)
bull Disadvantagesbull One must know the sequences to design the arraybull Even if one knows the sequences one cannot fit all of
them in a small number of arraysbull High noise level due to cross-hybrization non-linearity
etc
Microarrays
bull cDNA arrays bull oligonucleotide arrays
PLATFORMS AND TECHNOLOGY
bull There are five leading instruments that can be classified as part of the NGS technologies
bull the 454 GS FLX bull the Ion Torrentbull the SOLiD bull the Illumina bull and the more recently released PacBio
instrument
NGS platform properties
bull NGS platforms can be distinguished from each other based on
bull the chemistry employed for sequencing bull the amount of sequence information
producedbull the length of each sequence read bull the overall price per nt
454 GENOME SEQUENCER-FLXTM
bull The 454 pyro sequencer manufactured by Rochebull Principlebull Pyro sequencing bull a method of detecting single nucleotide addition
by capturing the emission of light produced from the release of the by-product pyrophosphate during the polymerization of the DNA molecule (Droege and Hill 2008 Rothberg and Leamon 2008)
Pyrosequencing Solid Phase method
1st MethodSolid Phase
Immobilized DNA 3 enzymes Wash step to remove nucleotides after each
addition
Pyrosequencing liquid phase method
2nd MethodLiquid Phase
3 enzymes + apyrase (nucleotide degradation enzyme)Eliminates need for washing step
bull In the well of a microtiter platebull primed DNA templatebull 4 enzymesbull Nucleotides are added stepwisebull Nucleotide-degrading enzyme degrade previous nucleotides
Pyrosequencing Method
Pyrosequencing Results
Example of NGS-BASED STUDIES
ION TORRENT SEMICONDUCTOR SEQUENCER
bull Modified version of the 454 pyrosequencing approach and operates
bull Based on the same sequencing chemistry except that it makes use of the H+ that is released with every nucleotide incorporated instead of the pyrophosphate (Rothberg et al2011)
bull Significantly reduces the cost of sequencing since luciferase and other costly enzymes and scanners are not needed
bull To date this instrument can sequence about 100nt but It should soon be able to read sequence lengths up to 200nt (IonTorrentSystemInc2)
SOLiD TM SYSTEM
bull The SOLiD genome sequencer from Applied Biosystems uses an emPCR process similar to 454 but parallel DNA sequencing is achieved by repeatedly ligating two-nucleotide probes instead of a sequencing reaction catalyzed by DNA polymerase (Morozova and Marra 2008)
bull The two-nucleotide probes are used to query adjacent bases on the DNA fragment therefore each nucleotide is actually probed twice
bull This system is designed to make sequence calls on two signals per base rather than one resulting in a lower error rate
bull Read approximately 35 nucleotides (Morozova and Marra 2008) but current versions of the instrument have increased the read-length to about 50 nucleotides (Applied Biosystems)
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
Overview Dideoxy (Sanger) Method
1
4
32
Gel electrophoresis
5
Dideoxy Method
bull Run four separate reactions each with different ddNTPsbull Run on a gel in four separate lanesbull Read the gel from the bottom up
Sanger sequencing vs NGS
bull Sanger sequencing is limiting because of the high cost and labor intensity
bull The development of next-generation sequencing (NGS) technologies has facilitated the collection of large amounts of nucleotide information in sequence read-length from 30 to 1500 nucleotides (nt) for hundreds of thousands to millions of DNA molecules simultaneously
bull In parallel the bioinformatics tools required to analyze these large datasets and identify unique gene sequences have also significantly improved
What is Next-Generation Sequencing
bull The advances of sequencing technologies have successfully contributed in elucidating the function of the human genome
bull NGS technologies have gained the capacity to sequence giga bases of DNA in a high-throughput and highly efficient manner that has not been possible using traditional Sanger sequencing
bull While Sanger is based on gel separation of chain-terminated fragments from enzymatic synthesis most NGS techniques are based on locally bound nano-clusters of template DNA and incorporation of fluorescent-labeled nucleotides by DNA polymerases or ligation processes
Microarrays
bull Extremely successfulbull Popular applications Gene expression profiling DNA
copy number (comparative genomic hybridization) SNPs microRNAs ChIP-chip (tiling arrays) splicing (exon arrays)
bull Disadvantagesbull One must know the sequences to design the arraybull Even if one knows the sequences one cannot fit all of
them in a small number of arraysbull High noise level due to cross-hybrization non-linearity
etc
Microarrays
bull cDNA arrays bull oligonucleotide arrays
PLATFORMS AND TECHNOLOGY
bull There are five leading instruments that can be classified as part of the NGS technologies
bull the 454 GS FLX bull the Ion Torrentbull the SOLiD bull the Illumina bull and the more recently released PacBio
instrument
NGS platform properties
bull NGS platforms can be distinguished from each other based on
bull the chemistry employed for sequencing bull the amount of sequence information
producedbull the length of each sequence read bull the overall price per nt
454 GENOME SEQUENCER-FLXTM
bull The 454 pyro sequencer manufactured by Rochebull Principlebull Pyro sequencing bull a method of detecting single nucleotide addition
by capturing the emission of light produced from the release of the by-product pyrophosphate during the polymerization of the DNA molecule (Droege and Hill 2008 Rothberg and Leamon 2008)
Pyrosequencing Solid Phase method
1st MethodSolid Phase
Immobilized DNA 3 enzymes Wash step to remove nucleotides after each
addition
Pyrosequencing liquid phase method
2nd MethodLiquid Phase
3 enzymes + apyrase (nucleotide degradation enzyme)Eliminates need for washing step
bull In the well of a microtiter platebull primed DNA templatebull 4 enzymesbull Nucleotides are added stepwisebull Nucleotide-degrading enzyme degrade previous nucleotides
Pyrosequencing Method
Pyrosequencing Results
Example of NGS-BASED STUDIES
ION TORRENT SEMICONDUCTOR SEQUENCER
bull Modified version of the 454 pyrosequencing approach and operates
bull Based on the same sequencing chemistry except that it makes use of the H+ that is released with every nucleotide incorporated instead of the pyrophosphate (Rothberg et al2011)
bull Significantly reduces the cost of sequencing since luciferase and other costly enzymes and scanners are not needed
bull To date this instrument can sequence about 100nt but It should soon be able to read sequence lengths up to 200nt (IonTorrentSystemInc2)
SOLiD TM SYSTEM
bull The SOLiD genome sequencer from Applied Biosystems uses an emPCR process similar to 454 but parallel DNA sequencing is achieved by repeatedly ligating two-nucleotide probes instead of a sequencing reaction catalyzed by DNA polymerase (Morozova and Marra 2008)
bull The two-nucleotide probes are used to query adjacent bases on the DNA fragment therefore each nucleotide is actually probed twice
bull This system is designed to make sequence calls on two signals per base rather than one resulting in a lower error rate
bull Read approximately 35 nucleotides (Morozova and Marra 2008) but current versions of the instrument have increased the read-length to about 50 nucleotides (Applied Biosystems)
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
Dideoxy Method
bull Run four separate reactions each with different ddNTPsbull Run on a gel in four separate lanesbull Read the gel from the bottom up
Sanger sequencing vs NGS
bull Sanger sequencing is limiting because of the high cost and labor intensity
bull The development of next-generation sequencing (NGS) technologies has facilitated the collection of large amounts of nucleotide information in sequence read-length from 30 to 1500 nucleotides (nt) for hundreds of thousands to millions of DNA molecules simultaneously
bull In parallel the bioinformatics tools required to analyze these large datasets and identify unique gene sequences have also significantly improved
What is Next-Generation Sequencing
bull The advances of sequencing technologies have successfully contributed in elucidating the function of the human genome
bull NGS technologies have gained the capacity to sequence giga bases of DNA in a high-throughput and highly efficient manner that has not been possible using traditional Sanger sequencing
bull While Sanger is based on gel separation of chain-terminated fragments from enzymatic synthesis most NGS techniques are based on locally bound nano-clusters of template DNA and incorporation of fluorescent-labeled nucleotides by DNA polymerases or ligation processes
Microarrays
bull Extremely successfulbull Popular applications Gene expression profiling DNA
copy number (comparative genomic hybridization) SNPs microRNAs ChIP-chip (tiling arrays) splicing (exon arrays)
bull Disadvantagesbull One must know the sequences to design the arraybull Even if one knows the sequences one cannot fit all of
them in a small number of arraysbull High noise level due to cross-hybrization non-linearity
etc
Microarrays
bull cDNA arrays bull oligonucleotide arrays
PLATFORMS AND TECHNOLOGY
bull There are five leading instruments that can be classified as part of the NGS technologies
bull the 454 GS FLX bull the Ion Torrentbull the SOLiD bull the Illumina bull and the more recently released PacBio
instrument
NGS platform properties
bull NGS platforms can be distinguished from each other based on
bull the chemistry employed for sequencing bull the amount of sequence information
producedbull the length of each sequence read bull the overall price per nt
454 GENOME SEQUENCER-FLXTM
bull The 454 pyro sequencer manufactured by Rochebull Principlebull Pyro sequencing bull a method of detecting single nucleotide addition
by capturing the emission of light produced from the release of the by-product pyrophosphate during the polymerization of the DNA molecule (Droege and Hill 2008 Rothberg and Leamon 2008)
Pyrosequencing Solid Phase method
1st MethodSolid Phase
Immobilized DNA 3 enzymes Wash step to remove nucleotides after each
addition
Pyrosequencing liquid phase method
2nd MethodLiquid Phase
3 enzymes + apyrase (nucleotide degradation enzyme)Eliminates need for washing step
bull In the well of a microtiter platebull primed DNA templatebull 4 enzymesbull Nucleotides are added stepwisebull Nucleotide-degrading enzyme degrade previous nucleotides
Pyrosequencing Method
Pyrosequencing Results
Example of NGS-BASED STUDIES
ION TORRENT SEMICONDUCTOR SEQUENCER
bull Modified version of the 454 pyrosequencing approach and operates
bull Based on the same sequencing chemistry except that it makes use of the H+ that is released with every nucleotide incorporated instead of the pyrophosphate (Rothberg et al2011)
bull Significantly reduces the cost of sequencing since luciferase and other costly enzymes and scanners are not needed
bull To date this instrument can sequence about 100nt but It should soon be able to read sequence lengths up to 200nt (IonTorrentSystemInc2)
SOLiD TM SYSTEM
bull The SOLiD genome sequencer from Applied Biosystems uses an emPCR process similar to 454 but parallel DNA sequencing is achieved by repeatedly ligating two-nucleotide probes instead of a sequencing reaction catalyzed by DNA polymerase (Morozova and Marra 2008)
bull The two-nucleotide probes are used to query adjacent bases on the DNA fragment therefore each nucleotide is actually probed twice
bull This system is designed to make sequence calls on two signals per base rather than one resulting in a lower error rate
bull Read approximately 35 nucleotides (Morozova and Marra 2008) but current versions of the instrument have increased the read-length to about 50 nucleotides (Applied Biosystems)
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
Sanger sequencing vs NGS
bull Sanger sequencing is limiting because of the high cost and labor intensity
bull The development of next-generation sequencing (NGS) technologies has facilitated the collection of large amounts of nucleotide information in sequence read-length from 30 to 1500 nucleotides (nt) for hundreds of thousands to millions of DNA molecules simultaneously
bull In parallel the bioinformatics tools required to analyze these large datasets and identify unique gene sequences have also significantly improved
What is Next-Generation Sequencing
bull The advances of sequencing technologies have successfully contributed in elucidating the function of the human genome
bull NGS technologies have gained the capacity to sequence giga bases of DNA in a high-throughput and highly efficient manner that has not been possible using traditional Sanger sequencing
bull While Sanger is based on gel separation of chain-terminated fragments from enzymatic synthesis most NGS techniques are based on locally bound nano-clusters of template DNA and incorporation of fluorescent-labeled nucleotides by DNA polymerases or ligation processes
Microarrays
bull Extremely successfulbull Popular applications Gene expression profiling DNA
copy number (comparative genomic hybridization) SNPs microRNAs ChIP-chip (tiling arrays) splicing (exon arrays)
bull Disadvantagesbull One must know the sequences to design the arraybull Even if one knows the sequences one cannot fit all of
them in a small number of arraysbull High noise level due to cross-hybrization non-linearity
etc
Microarrays
bull cDNA arrays bull oligonucleotide arrays
PLATFORMS AND TECHNOLOGY
bull There are five leading instruments that can be classified as part of the NGS technologies
bull the 454 GS FLX bull the Ion Torrentbull the SOLiD bull the Illumina bull and the more recently released PacBio
instrument
NGS platform properties
bull NGS platforms can be distinguished from each other based on
bull the chemistry employed for sequencing bull the amount of sequence information
producedbull the length of each sequence read bull the overall price per nt
454 GENOME SEQUENCER-FLXTM
bull The 454 pyro sequencer manufactured by Rochebull Principlebull Pyro sequencing bull a method of detecting single nucleotide addition
by capturing the emission of light produced from the release of the by-product pyrophosphate during the polymerization of the DNA molecule (Droege and Hill 2008 Rothberg and Leamon 2008)
Pyrosequencing Solid Phase method
1st MethodSolid Phase
Immobilized DNA 3 enzymes Wash step to remove nucleotides after each
addition
Pyrosequencing liquid phase method
2nd MethodLiquid Phase
3 enzymes + apyrase (nucleotide degradation enzyme)Eliminates need for washing step
bull In the well of a microtiter platebull primed DNA templatebull 4 enzymesbull Nucleotides are added stepwisebull Nucleotide-degrading enzyme degrade previous nucleotides
Pyrosequencing Method
Pyrosequencing Results
Example of NGS-BASED STUDIES
ION TORRENT SEMICONDUCTOR SEQUENCER
bull Modified version of the 454 pyrosequencing approach and operates
bull Based on the same sequencing chemistry except that it makes use of the H+ that is released with every nucleotide incorporated instead of the pyrophosphate (Rothberg et al2011)
bull Significantly reduces the cost of sequencing since luciferase and other costly enzymes and scanners are not needed
bull To date this instrument can sequence about 100nt but It should soon be able to read sequence lengths up to 200nt (IonTorrentSystemInc2)
SOLiD TM SYSTEM
bull The SOLiD genome sequencer from Applied Biosystems uses an emPCR process similar to 454 but parallel DNA sequencing is achieved by repeatedly ligating two-nucleotide probes instead of a sequencing reaction catalyzed by DNA polymerase (Morozova and Marra 2008)
bull The two-nucleotide probes are used to query adjacent bases on the DNA fragment therefore each nucleotide is actually probed twice
bull This system is designed to make sequence calls on two signals per base rather than one resulting in a lower error rate
bull Read approximately 35 nucleotides (Morozova and Marra 2008) but current versions of the instrument have increased the read-length to about 50 nucleotides (Applied Biosystems)
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
What is Next-Generation Sequencing
bull The advances of sequencing technologies have successfully contributed in elucidating the function of the human genome
bull NGS technologies have gained the capacity to sequence giga bases of DNA in a high-throughput and highly efficient manner that has not been possible using traditional Sanger sequencing
bull While Sanger is based on gel separation of chain-terminated fragments from enzymatic synthesis most NGS techniques are based on locally bound nano-clusters of template DNA and incorporation of fluorescent-labeled nucleotides by DNA polymerases or ligation processes
Microarrays
bull Extremely successfulbull Popular applications Gene expression profiling DNA
copy number (comparative genomic hybridization) SNPs microRNAs ChIP-chip (tiling arrays) splicing (exon arrays)
bull Disadvantagesbull One must know the sequences to design the arraybull Even if one knows the sequences one cannot fit all of
them in a small number of arraysbull High noise level due to cross-hybrization non-linearity
etc
Microarrays
bull cDNA arrays bull oligonucleotide arrays
PLATFORMS AND TECHNOLOGY
bull There are five leading instruments that can be classified as part of the NGS technologies
bull the 454 GS FLX bull the Ion Torrentbull the SOLiD bull the Illumina bull and the more recently released PacBio
instrument
NGS platform properties
bull NGS platforms can be distinguished from each other based on
bull the chemistry employed for sequencing bull the amount of sequence information
producedbull the length of each sequence read bull the overall price per nt
454 GENOME SEQUENCER-FLXTM
bull The 454 pyro sequencer manufactured by Rochebull Principlebull Pyro sequencing bull a method of detecting single nucleotide addition
by capturing the emission of light produced from the release of the by-product pyrophosphate during the polymerization of the DNA molecule (Droege and Hill 2008 Rothberg and Leamon 2008)
Pyrosequencing Solid Phase method
1st MethodSolid Phase
Immobilized DNA 3 enzymes Wash step to remove nucleotides after each
addition
Pyrosequencing liquid phase method
2nd MethodLiquid Phase
3 enzymes + apyrase (nucleotide degradation enzyme)Eliminates need for washing step
bull In the well of a microtiter platebull primed DNA templatebull 4 enzymesbull Nucleotides are added stepwisebull Nucleotide-degrading enzyme degrade previous nucleotides
Pyrosequencing Method
Pyrosequencing Results
Example of NGS-BASED STUDIES
ION TORRENT SEMICONDUCTOR SEQUENCER
bull Modified version of the 454 pyrosequencing approach and operates
bull Based on the same sequencing chemistry except that it makes use of the H+ that is released with every nucleotide incorporated instead of the pyrophosphate (Rothberg et al2011)
bull Significantly reduces the cost of sequencing since luciferase and other costly enzymes and scanners are not needed
bull To date this instrument can sequence about 100nt but It should soon be able to read sequence lengths up to 200nt (IonTorrentSystemInc2)
SOLiD TM SYSTEM
bull The SOLiD genome sequencer from Applied Biosystems uses an emPCR process similar to 454 but parallel DNA sequencing is achieved by repeatedly ligating two-nucleotide probes instead of a sequencing reaction catalyzed by DNA polymerase (Morozova and Marra 2008)
bull The two-nucleotide probes are used to query adjacent bases on the DNA fragment therefore each nucleotide is actually probed twice
bull This system is designed to make sequence calls on two signals per base rather than one resulting in a lower error rate
bull Read approximately 35 nucleotides (Morozova and Marra 2008) but current versions of the instrument have increased the read-length to about 50 nucleotides (Applied Biosystems)
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
Microarrays
bull Extremely successfulbull Popular applications Gene expression profiling DNA
copy number (comparative genomic hybridization) SNPs microRNAs ChIP-chip (tiling arrays) splicing (exon arrays)
bull Disadvantagesbull One must know the sequences to design the arraybull Even if one knows the sequences one cannot fit all of
them in a small number of arraysbull High noise level due to cross-hybrization non-linearity
etc
Microarrays
bull cDNA arrays bull oligonucleotide arrays
PLATFORMS AND TECHNOLOGY
bull There are five leading instruments that can be classified as part of the NGS technologies
bull the 454 GS FLX bull the Ion Torrentbull the SOLiD bull the Illumina bull and the more recently released PacBio
instrument
NGS platform properties
bull NGS platforms can be distinguished from each other based on
bull the chemistry employed for sequencing bull the amount of sequence information
producedbull the length of each sequence read bull the overall price per nt
454 GENOME SEQUENCER-FLXTM
bull The 454 pyro sequencer manufactured by Rochebull Principlebull Pyro sequencing bull a method of detecting single nucleotide addition
by capturing the emission of light produced from the release of the by-product pyrophosphate during the polymerization of the DNA molecule (Droege and Hill 2008 Rothberg and Leamon 2008)
Pyrosequencing Solid Phase method
1st MethodSolid Phase
Immobilized DNA 3 enzymes Wash step to remove nucleotides after each
addition
Pyrosequencing liquid phase method
2nd MethodLiquid Phase
3 enzymes + apyrase (nucleotide degradation enzyme)Eliminates need for washing step
bull In the well of a microtiter platebull primed DNA templatebull 4 enzymesbull Nucleotides are added stepwisebull Nucleotide-degrading enzyme degrade previous nucleotides
Pyrosequencing Method
Pyrosequencing Results
Example of NGS-BASED STUDIES
ION TORRENT SEMICONDUCTOR SEQUENCER
bull Modified version of the 454 pyrosequencing approach and operates
bull Based on the same sequencing chemistry except that it makes use of the H+ that is released with every nucleotide incorporated instead of the pyrophosphate (Rothberg et al2011)
bull Significantly reduces the cost of sequencing since luciferase and other costly enzymes and scanners are not needed
bull To date this instrument can sequence about 100nt but It should soon be able to read sequence lengths up to 200nt (IonTorrentSystemInc2)
SOLiD TM SYSTEM
bull The SOLiD genome sequencer from Applied Biosystems uses an emPCR process similar to 454 but parallel DNA sequencing is achieved by repeatedly ligating two-nucleotide probes instead of a sequencing reaction catalyzed by DNA polymerase (Morozova and Marra 2008)
bull The two-nucleotide probes are used to query adjacent bases on the DNA fragment therefore each nucleotide is actually probed twice
bull This system is designed to make sequence calls on two signals per base rather than one resulting in a lower error rate
bull Read approximately 35 nucleotides (Morozova and Marra 2008) but current versions of the instrument have increased the read-length to about 50 nucleotides (Applied Biosystems)
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
Microarrays
bull cDNA arrays bull oligonucleotide arrays
PLATFORMS AND TECHNOLOGY
bull There are five leading instruments that can be classified as part of the NGS technologies
bull the 454 GS FLX bull the Ion Torrentbull the SOLiD bull the Illumina bull and the more recently released PacBio
instrument
NGS platform properties
bull NGS platforms can be distinguished from each other based on
bull the chemistry employed for sequencing bull the amount of sequence information
producedbull the length of each sequence read bull the overall price per nt
454 GENOME SEQUENCER-FLXTM
bull The 454 pyro sequencer manufactured by Rochebull Principlebull Pyro sequencing bull a method of detecting single nucleotide addition
by capturing the emission of light produced from the release of the by-product pyrophosphate during the polymerization of the DNA molecule (Droege and Hill 2008 Rothberg and Leamon 2008)
Pyrosequencing Solid Phase method
1st MethodSolid Phase
Immobilized DNA 3 enzymes Wash step to remove nucleotides after each
addition
Pyrosequencing liquid phase method
2nd MethodLiquid Phase
3 enzymes + apyrase (nucleotide degradation enzyme)Eliminates need for washing step
bull In the well of a microtiter platebull primed DNA templatebull 4 enzymesbull Nucleotides are added stepwisebull Nucleotide-degrading enzyme degrade previous nucleotides
Pyrosequencing Method
Pyrosequencing Results
Example of NGS-BASED STUDIES
ION TORRENT SEMICONDUCTOR SEQUENCER
bull Modified version of the 454 pyrosequencing approach and operates
bull Based on the same sequencing chemistry except that it makes use of the H+ that is released with every nucleotide incorporated instead of the pyrophosphate (Rothberg et al2011)
bull Significantly reduces the cost of sequencing since luciferase and other costly enzymes and scanners are not needed
bull To date this instrument can sequence about 100nt but It should soon be able to read sequence lengths up to 200nt (IonTorrentSystemInc2)
SOLiD TM SYSTEM
bull The SOLiD genome sequencer from Applied Biosystems uses an emPCR process similar to 454 but parallel DNA sequencing is achieved by repeatedly ligating two-nucleotide probes instead of a sequencing reaction catalyzed by DNA polymerase (Morozova and Marra 2008)
bull The two-nucleotide probes are used to query adjacent bases on the DNA fragment therefore each nucleotide is actually probed twice
bull This system is designed to make sequence calls on two signals per base rather than one resulting in a lower error rate
bull Read approximately 35 nucleotides (Morozova and Marra 2008) but current versions of the instrument have increased the read-length to about 50 nucleotides (Applied Biosystems)
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
PLATFORMS AND TECHNOLOGY
bull There are five leading instruments that can be classified as part of the NGS technologies
bull the 454 GS FLX bull the Ion Torrentbull the SOLiD bull the Illumina bull and the more recently released PacBio
instrument
NGS platform properties
bull NGS platforms can be distinguished from each other based on
bull the chemistry employed for sequencing bull the amount of sequence information
producedbull the length of each sequence read bull the overall price per nt
454 GENOME SEQUENCER-FLXTM
bull The 454 pyro sequencer manufactured by Rochebull Principlebull Pyro sequencing bull a method of detecting single nucleotide addition
by capturing the emission of light produced from the release of the by-product pyrophosphate during the polymerization of the DNA molecule (Droege and Hill 2008 Rothberg and Leamon 2008)
Pyrosequencing Solid Phase method
1st MethodSolid Phase
Immobilized DNA 3 enzymes Wash step to remove nucleotides after each
addition
Pyrosequencing liquid phase method
2nd MethodLiquid Phase
3 enzymes + apyrase (nucleotide degradation enzyme)Eliminates need for washing step
bull In the well of a microtiter platebull primed DNA templatebull 4 enzymesbull Nucleotides are added stepwisebull Nucleotide-degrading enzyme degrade previous nucleotides
Pyrosequencing Method
Pyrosequencing Results
Example of NGS-BASED STUDIES
ION TORRENT SEMICONDUCTOR SEQUENCER
bull Modified version of the 454 pyrosequencing approach and operates
bull Based on the same sequencing chemistry except that it makes use of the H+ that is released with every nucleotide incorporated instead of the pyrophosphate (Rothberg et al2011)
bull Significantly reduces the cost of sequencing since luciferase and other costly enzymes and scanners are not needed
bull To date this instrument can sequence about 100nt but It should soon be able to read sequence lengths up to 200nt (IonTorrentSystemInc2)
SOLiD TM SYSTEM
bull The SOLiD genome sequencer from Applied Biosystems uses an emPCR process similar to 454 but parallel DNA sequencing is achieved by repeatedly ligating two-nucleotide probes instead of a sequencing reaction catalyzed by DNA polymerase (Morozova and Marra 2008)
bull The two-nucleotide probes are used to query adjacent bases on the DNA fragment therefore each nucleotide is actually probed twice
bull This system is designed to make sequence calls on two signals per base rather than one resulting in a lower error rate
bull Read approximately 35 nucleotides (Morozova and Marra 2008) but current versions of the instrument have increased the read-length to about 50 nucleotides (Applied Biosystems)
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
NGS platform properties
bull NGS platforms can be distinguished from each other based on
bull the chemistry employed for sequencing bull the amount of sequence information
producedbull the length of each sequence read bull the overall price per nt
454 GENOME SEQUENCER-FLXTM
bull The 454 pyro sequencer manufactured by Rochebull Principlebull Pyro sequencing bull a method of detecting single nucleotide addition
by capturing the emission of light produced from the release of the by-product pyrophosphate during the polymerization of the DNA molecule (Droege and Hill 2008 Rothberg and Leamon 2008)
Pyrosequencing Solid Phase method
1st MethodSolid Phase
Immobilized DNA 3 enzymes Wash step to remove nucleotides after each
addition
Pyrosequencing liquid phase method
2nd MethodLiquid Phase
3 enzymes + apyrase (nucleotide degradation enzyme)Eliminates need for washing step
bull In the well of a microtiter platebull primed DNA templatebull 4 enzymesbull Nucleotides are added stepwisebull Nucleotide-degrading enzyme degrade previous nucleotides
Pyrosequencing Method
Pyrosequencing Results
Example of NGS-BASED STUDIES
ION TORRENT SEMICONDUCTOR SEQUENCER
bull Modified version of the 454 pyrosequencing approach and operates
bull Based on the same sequencing chemistry except that it makes use of the H+ that is released with every nucleotide incorporated instead of the pyrophosphate (Rothberg et al2011)
bull Significantly reduces the cost of sequencing since luciferase and other costly enzymes and scanners are not needed
bull To date this instrument can sequence about 100nt but It should soon be able to read sequence lengths up to 200nt (IonTorrentSystemInc2)
SOLiD TM SYSTEM
bull The SOLiD genome sequencer from Applied Biosystems uses an emPCR process similar to 454 but parallel DNA sequencing is achieved by repeatedly ligating two-nucleotide probes instead of a sequencing reaction catalyzed by DNA polymerase (Morozova and Marra 2008)
bull The two-nucleotide probes are used to query adjacent bases on the DNA fragment therefore each nucleotide is actually probed twice
bull This system is designed to make sequence calls on two signals per base rather than one resulting in a lower error rate
bull Read approximately 35 nucleotides (Morozova and Marra 2008) but current versions of the instrument have increased the read-length to about 50 nucleotides (Applied Biosystems)
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
454 GENOME SEQUENCER-FLXTM
bull The 454 pyro sequencer manufactured by Rochebull Principlebull Pyro sequencing bull a method of detecting single nucleotide addition
by capturing the emission of light produced from the release of the by-product pyrophosphate during the polymerization of the DNA molecule (Droege and Hill 2008 Rothberg and Leamon 2008)
Pyrosequencing Solid Phase method
1st MethodSolid Phase
Immobilized DNA 3 enzymes Wash step to remove nucleotides after each
addition
Pyrosequencing liquid phase method
2nd MethodLiquid Phase
3 enzymes + apyrase (nucleotide degradation enzyme)Eliminates need for washing step
bull In the well of a microtiter platebull primed DNA templatebull 4 enzymesbull Nucleotides are added stepwisebull Nucleotide-degrading enzyme degrade previous nucleotides
Pyrosequencing Method
Pyrosequencing Results
Example of NGS-BASED STUDIES
ION TORRENT SEMICONDUCTOR SEQUENCER
bull Modified version of the 454 pyrosequencing approach and operates
bull Based on the same sequencing chemistry except that it makes use of the H+ that is released with every nucleotide incorporated instead of the pyrophosphate (Rothberg et al2011)
bull Significantly reduces the cost of sequencing since luciferase and other costly enzymes and scanners are not needed
bull To date this instrument can sequence about 100nt but It should soon be able to read sequence lengths up to 200nt (IonTorrentSystemInc2)
SOLiD TM SYSTEM
bull The SOLiD genome sequencer from Applied Biosystems uses an emPCR process similar to 454 but parallel DNA sequencing is achieved by repeatedly ligating two-nucleotide probes instead of a sequencing reaction catalyzed by DNA polymerase (Morozova and Marra 2008)
bull The two-nucleotide probes are used to query adjacent bases on the DNA fragment therefore each nucleotide is actually probed twice
bull This system is designed to make sequence calls on two signals per base rather than one resulting in a lower error rate
bull Read approximately 35 nucleotides (Morozova and Marra 2008) but current versions of the instrument have increased the read-length to about 50 nucleotides (Applied Biosystems)
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
Pyrosequencing Solid Phase method
1st MethodSolid Phase
Immobilized DNA 3 enzymes Wash step to remove nucleotides after each
addition
Pyrosequencing liquid phase method
2nd MethodLiquid Phase
3 enzymes + apyrase (nucleotide degradation enzyme)Eliminates need for washing step
bull In the well of a microtiter platebull primed DNA templatebull 4 enzymesbull Nucleotides are added stepwisebull Nucleotide-degrading enzyme degrade previous nucleotides
Pyrosequencing Method
Pyrosequencing Results
Example of NGS-BASED STUDIES
ION TORRENT SEMICONDUCTOR SEQUENCER
bull Modified version of the 454 pyrosequencing approach and operates
bull Based on the same sequencing chemistry except that it makes use of the H+ that is released with every nucleotide incorporated instead of the pyrophosphate (Rothberg et al2011)
bull Significantly reduces the cost of sequencing since luciferase and other costly enzymes and scanners are not needed
bull To date this instrument can sequence about 100nt but It should soon be able to read sequence lengths up to 200nt (IonTorrentSystemInc2)
SOLiD TM SYSTEM
bull The SOLiD genome sequencer from Applied Biosystems uses an emPCR process similar to 454 but parallel DNA sequencing is achieved by repeatedly ligating two-nucleotide probes instead of a sequencing reaction catalyzed by DNA polymerase (Morozova and Marra 2008)
bull The two-nucleotide probes are used to query adjacent bases on the DNA fragment therefore each nucleotide is actually probed twice
bull This system is designed to make sequence calls on two signals per base rather than one resulting in a lower error rate
bull Read approximately 35 nucleotides (Morozova and Marra 2008) but current versions of the instrument have increased the read-length to about 50 nucleotides (Applied Biosystems)
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
Pyrosequencing liquid phase method
2nd MethodLiquid Phase
3 enzymes + apyrase (nucleotide degradation enzyme)Eliminates need for washing step
bull In the well of a microtiter platebull primed DNA templatebull 4 enzymesbull Nucleotides are added stepwisebull Nucleotide-degrading enzyme degrade previous nucleotides
Pyrosequencing Method
Pyrosequencing Results
Example of NGS-BASED STUDIES
ION TORRENT SEMICONDUCTOR SEQUENCER
bull Modified version of the 454 pyrosequencing approach and operates
bull Based on the same sequencing chemistry except that it makes use of the H+ that is released with every nucleotide incorporated instead of the pyrophosphate (Rothberg et al2011)
bull Significantly reduces the cost of sequencing since luciferase and other costly enzymes and scanners are not needed
bull To date this instrument can sequence about 100nt but It should soon be able to read sequence lengths up to 200nt (IonTorrentSystemInc2)
SOLiD TM SYSTEM
bull The SOLiD genome sequencer from Applied Biosystems uses an emPCR process similar to 454 but parallel DNA sequencing is achieved by repeatedly ligating two-nucleotide probes instead of a sequencing reaction catalyzed by DNA polymerase (Morozova and Marra 2008)
bull The two-nucleotide probes are used to query adjacent bases on the DNA fragment therefore each nucleotide is actually probed twice
bull This system is designed to make sequence calls on two signals per base rather than one resulting in a lower error rate
bull Read approximately 35 nucleotides (Morozova and Marra 2008) but current versions of the instrument have increased the read-length to about 50 nucleotides (Applied Biosystems)
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
Pyrosequencing Method
Pyrosequencing Results
Example of NGS-BASED STUDIES
ION TORRENT SEMICONDUCTOR SEQUENCER
bull Modified version of the 454 pyrosequencing approach and operates
bull Based on the same sequencing chemistry except that it makes use of the H+ that is released with every nucleotide incorporated instead of the pyrophosphate (Rothberg et al2011)
bull Significantly reduces the cost of sequencing since luciferase and other costly enzymes and scanners are not needed
bull To date this instrument can sequence about 100nt but It should soon be able to read sequence lengths up to 200nt (IonTorrentSystemInc2)
SOLiD TM SYSTEM
bull The SOLiD genome sequencer from Applied Biosystems uses an emPCR process similar to 454 but parallel DNA sequencing is achieved by repeatedly ligating two-nucleotide probes instead of a sequencing reaction catalyzed by DNA polymerase (Morozova and Marra 2008)
bull The two-nucleotide probes are used to query adjacent bases on the DNA fragment therefore each nucleotide is actually probed twice
bull This system is designed to make sequence calls on two signals per base rather than one resulting in a lower error rate
bull Read approximately 35 nucleotides (Morozova and Marra 2008) but current versions of the instrument have increased the read-length to about 50 nucleotides (Applied Biosystems)
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
Pyrosequencing Results
Example of NGS-BASED STUDIES
ION TORRENT SEMICONDUCTOR SEQUENCER
bull Modified version of the 454 pyrosequencing approach and operates
bull Based on the same sequencing chemistry except that it makes use of the H+ that is released with every nucleotide incorporated instead of the pyrophosphate (Rothberg et al2011)
bull Significantly reduces the cost of sequencing since luciferase and other costly enzymes and scanners are not needed
bull To date this instrument can sequence about 100nt but It should soon be able to read sequence lengths up to 200nt (IonTorrentSystemInc2)
SOLiD TM SYSTEM
bull The SOLiD genome sequencer from Applied Biosystems uses an emPCR process similar to 454 but parallel DNA sequencing is achieved by repeatedly ligating two-nucleotide probes instead of a sequencing reaction catalyzed by DNA polymerase (Morozova and Marra 2008)
bull The two-nucleotide probes are used to query adjacent bases on the DNA fragment therefore each nucleotide is actually probed twice
bull This system is designed to make sequence calls on two signals per base rather than one resulting in a lower error rate
bull Read approximately 35 nucleotides (Morozova and Marra 2008) but current versions of the instrument have increased the read-length to about 50 nucleotides (Applied Biosystems)
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
Example of NGS-BASED STUDIES
ION TORRENT SEMICONDUCTOR SEQUENCER
bull Modified version of the 454 pyrosequencing approach and operates
bull Based on the same sequencing chemistry except that it makes use of the H+ that is released with every nucleotide incorporated instead of the pyrophosphate (Rothberg et al2011)
bull Significantly reduces the cost of sequencing since luciferase and other costly enzymes and scanners are not needed
bull To date this instrument can sequence about 100nt but It should soon be able to read sequence lengths up to 200nt (IonTorrentSystemInc2)
SOLiD TM SYSTEM
bull The SOLiD genome sequencer from Applied Biosystems uses an emPCR process similar to 454 but parallel DNA sequencing is achieved by repeatedly ligating two-nucleotide probes instead of a sequencing reaction catalyzed by DNA polymerase (Morozova and Marra 2008)
bull The two-nucleotide probes are used to query adjacent bases on the DNA fragment therefore each nucleotide is actually probed twice
bull This system is designed to make sequence calls on two signals per base rather than one resulting in a lower error rate
bull Read approximately 35 nucleotides (Morozova and Marra 2008) but current versions of the instrument have increased the read-length to about 50 nucleotides (Applied Biosystems)
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
ION TORRENT SEMICONDUCTOR SEQUENCER
bull Modified version of the 454 pyrosequencing approach and operates
bull Based on the same sequencing chemistry except that it makes use of the H+ that is released with every nucleotide incorporated instead of the pyrophosphate (Rothberg et al2011)
bull Significantly reduces the cost of sequencing since luciferase and other costly enzymes and scanners are not needed
bull To date this instrument can sequence about 100nt but It should soon be able to read sequence lengths up to 200nt (IonTorrentSystemInc2)
SOLiD TM SYSTEM
bull The SOLiD genome sequencer from Applied Biosystems uses an emPCR process similar to 454 but parallel DNA sequencing is achieved by repeatedly ligating two-nucleotide probes instead of a sequencing reaction catalyzed by DNA polymerase (Morozova and Marra 2008)
bull The two-nucleotide probes are used to query adjacent bases on the DNA fragment therefore each nucleotide is actually probed twice
bull This system is designed to make sequence calls on two signals per base rather than one resulting in a lower error rate
bull Read approximately 35 nucleotides (Morozova and Marra 2008) but current versions of the instrument have increased the read-length to about 50 nucleotides (Applied Biosystems)
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
SOLiD TM SYSTEM
bull The SOLiD genome sequencer from Applied Biosystems uses an emPCR process similar to 454 but parallel DNA sequencing is achieved by repeatedly ligating two-nucleotide probes instead of a sequencing reaction catalyzed by DNA polymerase (Morozova and Marra 2008)
bull The two-nucleotide probes are used to query adjacent bases on the DNA fragment therefore each nucleotide is actually probed twice
bull This system is designed to make sequence calls on two signals per base rather than one resulting in a lower error rate
bull Read approximately 35 nucleotides (Morozova and Marra 2008) but current versions of the instrument have increased the read-length to about 50 nucleotides (Applied Biosystems)
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
ILLUMINA GENOME ANALYZER bull The Illumina Solexa technology bull Differs from 454 and SOLiD in terms of its amplification strategybull Rather than amplifying DNA-covered beads by emPCR the Illumina
technology amplifies clusters of DNA fragments that are affixed to a glass slide using a strategy called bridge amplification
bull The parallel sequencing process uses dye-labeled nucleotides (one fluoro phore per base) that are added simultaneously rather than sequentially as in the 454 process
bull The DNA clusters are then subjected to laser excitation that cleaves the dye and permits the addition of the next nucleotide
bull In 2008 Illumina sequencer projects reported reads of 25ndash50 ntbull Currently the Illumina sequencer can produce longer reads of 100nt
(Illumina Inc)
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
PacBio RS bull PacBio is a single-molecule sequencing approach bull PacBio has been developed to further reduce the cost and time required to
obtain the sequence of a genome or transcriptomebull It is thought of as a ldquothird generationrdquo sequencing platformbull The PacBio works based on a nanophotonic tool called zero-mode waveguide
(ZMW Levene et al 2003)bull ZMW technology allows for a DNA polymerase to work in real time using
fluorescently labeled nucleotides and tracks synthesis of a single molecule per DNA fragment (Eid et al 2009)
bull Like the 454 and Illumina instruments the PacBio sequences by measuring the burst of light produced when the pyrophosphate and fluorescent label are released during the polymerization reaction
bull This instrument is able to sequence single molecules up to1500nt long but the error rate (around 15) is still relatively high (Pacific Biosciences)
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
conclusion
bull As the costs for DNA and RNA sequencing decrease the combination of several NGS platforms should facilitate whole genome sequencing projects and expand our knowledge of ecologically relevant species
bull Under- standing the relationships between environmental chemical exposure and gene expression will provide valuable data for environmental risk assessments (ERA)
bull The introduction of NGS technologies has tremendously changed the landscape of genomic research
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
FUTURE PERSPECTIVE
bull NGS-Towards Personalized Medicinebull It will be necessary to integrate the extensive
genomic data gathered from transcriptomics gene regulation and evolutionary biology into a working framework in order to propose new hypotheses in life sciences
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
referencesbull Dunham I Kundaje A Aldred SF Collins PJ Davis CA Doyle F Epstein CBbull Frietze S Harrow J Kaul R et al An integrated encyclopedia of DNA elements in thebull human genome Nature 2012 489 57104875374bull 2 Sastre L Clinical implications of the encode project Clin Transl Oncol 2012 14 8011048753802bull 3 Frazer KA Decoding the human genome Genome Res 2012 22 159910487531601bull 4 Database of Genomic Variants Available online httpprojectstcagca (accessed on 5 July
2012)bull 5 Ecker JR Bickmore WA Barroso I Pritchard JK Gilad Y Segal E Genomics Encodebull explained Nature 2012 489 52104875355bull 6 Sanger F Nicklen S Coulson AR DNA sequencing with chain-terminating inhibitors Procbull Natl Acad Sci USA 1977 74 546310487535467bull 7 Sanger F Coulson AR A rapid method for determining sequences in DNA by primedbull synthesis with DNA polymerase J Mol Biol 1975 94 4411048753448bull DouglasSE(2006)Microarraystud- ies ofgeneexpressioninfish OMICS 10 474ndash489bull DroegeMandHillB(2008)The Genome SequencerFLXTM System ndash
longerreadsmoreapplications straightforwardbioinformaticsand morecompletedatasets J Biotech- nol 136 3ndash10
