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Next-generation-sequencingBasic principles and sequencing platforms
WIV-ISP workshop 10/10/2014
Annelies Haegeman
Institute for Agricultural and Fisheries ResearchPlant Sciences Unit
www.ilvo.vlaanderen.be
Agriculture and Fisheries Policy Area
10/10/2014 What is next generation sequencing? 2
Presentation outline
• Sanger sequencing (1977 – present)
• Next generation sequencing (2005 – present)• 454
• Ion Torrent
• Illumina
• 3rd generation sequencing (2012 – present)• PacBio or SMRT sequencing
• Oxford Nanopore
• Sequencing @ ILVO
What is next generation sequencing? 3
NGS experiment setup
DNA sample NGS Instrument Data
Library Preparation
Sequencing Data Analysis
10/10/2014
What is next generation sequencing? 4
Presentation outline
• Sanger sequencing (1977 – present)
• Next generation sequencing (2005 – present)• 454
• Ion Torrent
• Illumina
• 3rd generation sequencing (2012 – present)• PacBio or SMRT sequencing
• Oxford Nanopore
• Sequencing @ ILVO
10/10/2014
What is next generation sequencing? 5
Sanger sequencing (1977)
10/10/2014
What is next generation sequencing? 6
Sanger sequencing
10/10/2014
http://en.wikipedia.org/wiki/Sanger_sequencing
What is next generation sequencing? 7
Applied Biosystems
• 1987 : First sequencer (ABI370)
• 1996 : First capillary sequencer (ABI 310)
• Present : ABI 3500
10/10/2014
What is next generation sequencing? 8
Presentation outline
• Sanger sequencing (1977 – present)
• Next generation sequencing (2005 – present)• 454
• Ion Torrent
• Illumina
• 3rd generation sequencing (2012 – present)• PacBio or SMRT sequencing
• Oxford Nanopore
• Sequencing @ ILVO
10/10/2014
What is next generation sequencing? 9
454 sequencing
• 2005
• DNA is fragmented, adapter ligated
and denatured
• Emulsion PCR
10/10/2014
Rothberg, J.M. & Leamon, J.H. Nature Biotechnology 26, 1117 - 1124
What is next generation sequencing? 10
454 sequencing
• Put beads and reagents in picotiter plate (one DNA bead
per well)
10/10/2014
What is next generation sequencing? 11
454 sequencing
• Sequencing
• One bead = one read
• Reads up to 700 bp
• 1 million reads / run
10/10/2014
What is next generation sequencing? 12
454 sequencing
GS Junior• ~ 400 bp reads
• Up to 100.000 reads / run
• 10h run time
GS FLX Titanium XL+• ~ 700 bp reads
• Up to 1 million reads/run
• 23h run time
10/10/2014
What is next generation sequencing? 13
454 sequencing
• Pros
• First commercially available NGS technique
• Relatively long reads
• Relatively short run time
• Cons
• Accuracy problems with homopolymeric repeats
• Emulsion PCR
• Relatively few reads
• Relatively expensive
• R.I.P. 2016
10/10/2014
What is next generation sequencing? 14
Presentation outline
• Sanger sequencing (1977 – present)
• Next generation sequencing (2005 – present)• 454
• Ion Torrent
• Illumina
• 3rd generation sequencing (2012 – present)• PacBio or SMRT sequencing
• Oxford Nanopore
• Sequencing @ ILVO
10/10/2014
• 2010, Life Technologies
• The first steps are similar to 454 sequencing
• Bind DNA to beads
• Emulsion PCR
• Chip with ion sensor
What is next generation sequencing? 15
Ion Torrent sequencing
10/10/2014
What is next generation sequencing? 16
Ion Torrent sequencing
• Semiconductor sequencing
10/10/2014
What is next generation sequencing? 17
Ion Torrent sequencing
• Semiconductor sequencing on a chip (no imaging)
10/10/2014
What is next generation sequencing? 18
Ion Torrent sequencing
Ion PGM• 3 types of chips
• 200 or 400 bp reads
• Up to 5.5 million reads / Ion 318 chip
• 4 – 7 h run time
Ion Proton• Up to 200 bp reads
• Up to 60-80 million reads
• 2 – 4 h run time
10/10/2014
What is next generation sequencing? 19
Ion Torrent sequencing
• Pros
• Fast
• Relatively cheap
• Scalable (different chips)
• Cons
• Relatively high error rate
• Emulsion PCR
• Ion isothermal amplification chemistry (November 2014)
• isothermal amplification instead of emulsion PCR
10/10/2014
What is next generation sequencing? 20
Presentation outline
• Sanger sequencing (1977 – present)
• Next generation sequencing (2005 – present)• 454
• Ion Torrent
• Illumina
• 3rd generation sequencing (2012 – present)• PacBio or SMRT sequencing
• Oxford Nanopore
• Sequencing @ ILVO
10/10/2014
What is next generation sequencing? 21
Illumina sequencing
• 2006 (Solexa)
• Most popular sequencing technique nowadays!
0
2000
4000
6000
8000
10000
12000
454 solid ion
torrent
illumina pacbio
# studies in NCBI SRA database (01/2014)
10/10/2014
10/10/2014 What is next generation sequencing? 22
Illumina sequencing
Metzker, Nature Reviews Genetics, 2010, 11, 31-46
What is next generation sequencing? 23
Paired end sequencing
10/10/2014
©Illumina
What is next generation sequencing? 24
Illumina sequencing
MiSeq• Up to 2 x 300 bp reads
• 15 million single reads
• 2.7 days run time
HiSeq2500• Up to 2 x 125 bp reads
• 2 billion single reads
• 6 days run time
10/10/2014
What is next generation sequencing? 25
Illumina sequencing
NextSeq 500• Up to 2 x 150bp reads
• Up to 400 million reads
• 1.2 days run time
HiSeq X Ten• 2 x 150 bp reads
• Up to 30 billion reads
• 3 days run time
10/10/2014
What is next generation sequencing? 26
Illumina sequencing
• Pros
• Very high throughput
• Best price/bp
• Cons
• Relatively long run time
• Sequence quality decreases towards the end
• Imaging interference in low diversity libraries
10/10/2014
What is next generation sequencing? 27
Advantages of NGS vs Sanger
• Cheap (cost per bp)• 1 bacterial genome (100x, 650Mb) < a 96-well plate (80 kb)
• (Very) high throughput (parallel sequencing)
• Multiplexing
10/10/2014
©Illumina
What is next generation sequencing? 28
How do I choose a platform?
• Read length vs. number of reads (read depth)
10/10/2014
What is next generation sequencing? 29
Overview
10/10/2014
What is next generation sequencing? 30
Overview
Platform ChemistryRead
LengthRun Time Reads/Run Advantage Disadvantage
454 GS JuniorPyro-
sequencing400 8 h 0.1 M
Long read
length
High error rate,
expensive
454 GS FLX+Pyro-
sequencing700 23 h 1 M
Long read
length
High error rate,
expensive
Ion PGMProton
Detection200-400 4-7 h 5.5 M Short run time
High error rate, but
will improve
Ion ProtonProton
Detection200 2-4 h 80 M Short run time
High error rate, but
will improve
Illumina MiSeqBridge
amplificationup to 2x300 2.7 days 25 M
High-
throughput /
cost
Long run time
Illumina HiSeqBridge
amplification2x125 6 days 2000 M
High-
throughput /
cost
Short reads, long run
time
10/10/2014
What is next generation sequencing? 31
Presentation outline
• Sanger sequencing (1977 – present)
• Next generation sequencing (2005 – present)• 454
• Illumina
• Ion Torrent
• 3rd generation sequencing (2011 – present)• PacBio or SMRT sequencing
• Oxford Nanopore
• Sequencing @ ILVO
PCR based
Single molecule
2nd
10/10/2014
What is next generation sequencing? 32
PacBio sequencing
• 2011, Pacific Biosciences
• Single Molecule Real Time Sequencing (SMRT)
10/10/2014
What is next generation sequencing? 33
PacBio sequencing
PacBio RSII• Up to 30 kb reads
• 50.000 reads
• Up to 3h run time
Often used in combination
with Illumina sequencing
10/10/2014
What is next generation sequencing? 34
PacBio sequencing
• Pros
• No PCR
• Very long reads
• No problems with high GC content
• Can detect nucleotide modifications (e.g. methylation)
• Cons
• High error rate, but expected to improve
10/10/2014
What is next generation sequencing? 35
Presentation outline
• Sanger sequencing (1977 – present)
• Next generation sequencing (2005 – present)• 454
• Illumina
• Ion Torrent
• 3rd generation sequencing (2011 – present)• PacBio or SMRT sequencing
• Oxford Nanopore
• Sequencing @ ILVO
PCR based
Single molecule
10/10/2014
What is next generation sequencing? 36
Oxford nanopore sequencing
10/10/2014
What is next generation sequencing? 37
Oxford nanopore sequencing
10/10/2014
• MinION portable sequencer
• Currently a beta version is being
tested in 500 selected labs
• Accuracy: 60-85%
• First reports on read lengths:
up to 79000 bases!
• Under development
• GridION
• PromethION
10/10/2014 What is next generation sequencing? 38
Presentation outline
• Sanger sequencing (1977 – present)
• Next generation sequencing (2005 – present)• 454
• Ion Torrent
• Illumina
• 3rd generation sequencing (2012 – present)• PacBio or SMRT sequencing
• Oxford Nanopore
• Sequencing @ ILVO
AnimalSciences
Cattle, Pig andSmall AnimalHusbandry
Fisheries andAquatic
Production
Marine Environment and Quality
Plant Sciences
CropProtection
Growth andDevelopment
Applied Genetics and
Breeding
CropHusbandry
Technology andFood Science
Agricultural Engineering
Product Quality andInnovation
Food Safety
Social Sciences
Agricultural and Farm
Development
RuralDevelopment
ILVO
What is next generation sequencing? 3910/10/2014
Plant Sciences
CropProtection
Growth andDevelopment
Applied Genetics and
Breeding
CropHusbandry
Sequencing @ ILVO
What is next generation sequencing? 4010/10/2014
Whole genome shotgun (WGS) sequencing
Plant pathogens, mainly bacteria
Amplicon sequencing (16S rRNA)
Bacterial populations in soil
Transcriptome sequencing
Azalea, perennial ryegrass
Targeted resequencing
Perennial ryegrass
Genotyping-by-sequencing (GBS)
Perennial ryegrass, Phytophthora oomycetes
Sequencing @ ILVO
What is next generation sequencing? 4110/10/2014
AnimalSciences
Cattle, Pig andSmall AnimalHusbandry
Marine Environment and Quality
Fisheries and
Aquatic
Production
Amplicon sequencing (16S rRNA)
Bacterial populations in:
- Cow rumen
- Marine environments (water, sediment, litter)
Whole genome shotgun sequencing(metagenomics)
- Pig gut (antibiotics resistance genes)
- Marine environments (plastic litter)
Genotyping-By-Sequencing (GBS)
Comb jelly
What is next generation sequencing? 42
Sequencing @ ILVO
www.ilvogenomics.be
10/10/2014
Questions?
WIV-ISP workshop 10/10/2014
Institute for Agricultural and Fisheries ResearchPlant Sciences Unit
www.ilvo.vlaanderen.be
Agriculture and Fisheries Policy Area