NGS technologies: a user s guide. Karim Gharbi & Mark Blaxter

Transcription

1 NGS technologies: a user s guide Karim Gharbi & Mark Blaxter genepool-manager@ed.ac.uk

2 Natural history of sequencing 2

3 Brief history of sequencing 100s bp throughput 100 Gb

4 4

5 NGS platforms Roche GS FLX+ Illumina GAIIx Illumina HiSeq 2000 SOLiD 4/5500 Read length Up to 1,000 bp (700) up to bp up to bp Up to 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 5

6 Instruments worldwide Instrument Total Illumina Genome Analyzer 526 Illumina HiSeq ABI SOLiD 328 Roche Pacific Biosciences 22 Polonator 9 6

7 Benchtop sequencers Roche 454 Junior Ion Torrent Illumina MiSeq Read length bp up to ~ 200 bp up to 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 7

8 Single-molecule sequencing PacBio RS Heliscope Nanopore GridIon Read length up to 1,000 bp bp? Throughput ~ 3 Gb Gb? No. reads ~ 3M 600M-1B? Run time ~ 2.5h 8 days? 8

9 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.. 9

10 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 10

11 Library preparation fragmentation adapter ligation (size selection) (barcoding/pcr amplification) 11

12 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 12

13 Illumina - solid-phase amplification Adapted from Metzker

14 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 14

15 Illumina - cyclic reversible termination Adapted from Metzker

16 Illumina - cyclic reversible termination Adapted from Metzker

17 Paired-end (PE) sequencing 17

18 Paired-end sequencing DNA 18

19 The NGS (expanding) toolkit marker discovery population genomics genome evolution biodiversity complex traits 19

20 Tips and best practice Key questions what is the question/hypothesis/objective? is next-generation sequencing the right tool? Common pitfalls technology/budget-driven experiments ( ) 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 20

21 Further reading Reviews Glenn TC. Molecular Ecology Resources 2011, 11: Ekblom R, Galindo J. Heredity 2010, 107:1 15 Metzker ML. Nature Reviews Genetics 2009, 11:31 46 Vendor s websites ( ) NextGenBUG The GenePool team