ngs technologies: a user's guide - botanical society of scotland
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Natural history of sequencing
Edinburgh, October 17-18, 2011RBGE NGS Workshop
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Brief history of sequencing
Edinburgh, October 17-18, 2011RBGE NGS Workshop
19771977 19861986 19951995 2005200519991999 20072007 20102010
100s bp 100 Gbthroughput
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http://pathogenomics.bham.ac.uk/hts/
Edinburgh, October 17-18, 2011RBGE NGS Workshop
NGS platforms
Roche GS FLX+ Illumina GAIIx Illumina HiSeq 2000 SOLiD 4/5500
Read length Up to 1,000 bp (700) up to 150 + 150 bp up to 100 + 100 bp Up to 75 + 35 bp
Throughput 700 Mb up to 95 Gb > 600 Gb 100 Gb
No. reads up to 1M up to 640M up to 6B up to 1.5B
Run time 23 hours up to 14 days up to 8 days up to 8 days
5Edinburgh, October 17-18, 2011RBGE NGS Workshop
Instruments worldwide
Instrument Total
Illumina Genome Analyzer 526
Illumina HiSeq 2000 463
ABI SOLiD 328
Roche 454 279
Pacific Biosciences 22
Polonator 9
http://pathogenomics.bham.ac.uk/hts/
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Benchtop sequencers
Roche 454 Junior Ion Torrent Illumina MiSeq
Read length 350-400 bp up to ~ 200 bp up to 150+150 bp
Throughput 35 Mb ~ 1 Gb up to 2 Gb
No. reads ~ 100,000 up to 11M up to 14M
Run time ~ 10h ~ 2h up to 27h
7Edinburgh, October 17-18, 2011RBGE NGS Workshop
Single-molecule sequencing
PacBio RS Heliscope Nanopore GridIon
Read length up to 1,000 bp 25-55 bp ?
Throughput ~ 3 Gb 21-35 Gb ?
No. reads ~ 3M 600M-1B ?
Run time ~ 2.5h 8 days ?
8Edinburgh, October 17-18, 2011RBGE NGS Workshop
NGS workflows
(fragmentation)
ligation
size selection
immobilisation
2h-7 days
clonal amplification
sequencing
2h-14 days
image processing
quality control
data analysis
usually more than you
think..
9Edinburgh, October 17-18, 2011RBGE NGS Workshop
NGS workflows
(fragmentation)
ligation
size selection
immobilisation
2h-7 days
clonal amplification
sequencing
2h-14 days
image processing
quality control
data analysis
usually more than you
think…
10Edinburgh, October 17-18, 2011RBGE NGS Workshop
Library preparation
fragmentation adapter ligation
(size selection)
(barcoding/PCR amplification)
11Edinburgh, October 17-18, 2011RBGE NGS Workshop
NGS workflows
(fragmentation)
ligation
size selection
immobilisation
2h-7 days
clonal amplification
sequencing
2h-14 days
image processing
quality control
data analysis
usually more than you
think…
12Edinburgh, October 17-18, 2011RBGE NGS Workshop
Illumina - solid-phase amplification
Adapted from Metzker 2010
13Edinburgh, October 17-18, 2011RBGE NGS Workshop
NGS workflows
(fragmentation)
ligation
size selection
immobilisation
2h-7 days
clonal amplification
sequencing
2h-14 days
image processing
quality control
data analysis
usually more than
you think…
14Edinburgh, October 17-18, 2011RBGE NGS Workshop
Illumina - cyclic reversible termination
Adapted from Metzker 2010
15Edinburgh, October 17-18, 2011RBGE NGS Workshop
Illumina - cyclic reversible termination
Adapted from Metzker 2010
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Paired-end (PE) sequencing
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Paired-end sequencing
DNA
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The NGS (expanding) toolkit
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marker discoverymarker discovery population
genomics
population
genomics biodiversitybiodiversitygenome
evolution
genome
evolution complex traitscomplex traits
Tips and best practice
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Key questions Common pitfalls
what is the question/hypothesis/objective? technology/budget-driven experiments (…)
is next-generation sequencing the right tool? a sledge hammer to crack a nut
which platform/technique is most appropriate? insufficient coverage, reads too short
what is the optimal design?
*sample size (replication)
*coverage per sample
*sequencing strategy
lack of replication
lack of coverage
“poor” reference genome/transcriptome
insufficient sample quality/quantity
what is the data analysis strategy? no/optimistic data analysis plan
Further reading…
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Reviews
Glenn TC. Molecular Ecology Resources 2011, 11:759–769
Ekblom R, Galindo J. Heredity 2010, 107:1–15
Metzker ML. Nature Reviews Genetics 2009, 11:31–46
Vendor’s websites (…)
http://seqanswers.com/
NextGenBUG
The GenePool team
http://genepool.bio.ed.ac.uk/
https://twitter.com/GenePoolNews