nd Generation (“NextGen”) Sequencing Technologies “Fantastic” bizarre or exotic; seeming more appropriate to a fairy tale than to reality or practical use.
Download ReportTranscript nd Generation (“NextGen”) Sequencing Technologies “Fantastic” bizarre or exotic; seeming more appropriate to a fairy tale than to reality or practical use.
nd 2 Generation (“NextGen”) Sequencing Technologies “Fantastic” bizarre or exotic; seeming more appropriate to a fairy tale than to reality or practical use % of Paired K-mers with Uniquely Assignable Location Read Length is Not As Important For Resequencing 100% 90% 80% 70% 60% E.COLI 50% HUMAN 40% 30% 20% 10% 0% 8 Jay Shendure 10 12 14 16 18 20 Length of K-mer Reads (bp) Paired End Reads are Important! Known Distance Read 1 Read 2 Repetitive DNA Unique DNA Paired read maps uniquely Single read maps to multiple positions emulsion PCR emPCR Margulies M et al., (2006) Genome sequencing in microfabricated high-density picolitre reactors Nature 437, 376-380 Roche 454 Margulies M et al., (2006) Genome sequencing in microfabricated high-density picolitre reactors Nature 437, 376-380 OH EE Slawson Tempel, © WUSTL OH EE Slawson Tempel, © WUSTL OH OH OH OH OH EE Slawson Tempel, © WUSTL OH OH EE Slawson Tempel, © WUSTL 3 2 1 EE Slawson Tempel, © WUSTL OH Pyrophosphate OH EE Slawson Tempel, © WUSTL OH EE Slawson Tempel, © WUSTL ATP + luciferin OH EE Slawson Tempel, © WUSTL ATP + luciferin OH EE Slawson Tempel, © WUSTL OH OH OH OH OH EE Slawson Tempel, © WUSTL OH OH EE Slawson Tempel, © WUSTL OH OH OH OH OH EE Slawson Tempel, © WUSTL OH OH OH OH EE Slawson Tempel, © WUSTL OH OH OH OH EE Slawson Tempel, © WUSTL OH OH OH EE Slawson Tempel, © WUSTL OH OH OH EE Slawson Tempel, © WUSTL OH EE Slawson Tempel, © WUSTL OH EE Slawson Tempel, © WUSTL ATP + luciferin ATP + luciferin ATP + luciferin OH EE Slawson Tempel, © WUSTL ATP + luciferin ATP + luciferin ATP + luciferin OH EE Slawson Tempel, © WUSTL Brightness of flash is proportional to number of nucleotides added Flash is too brigh Flash brightness 4-mer 3-mer 2-mer 1mer TCACTTCAAGGGT… EE Slawson Tempel, © WUSTL ~ 0.5 Gb/run Roche 454 Read length 350-400 bp 200 cycles A EE Slawson Tempel, © WUSTL T G C Illumina ~ 400 bp Nebulizer EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL Flow cell 8 channels (“lanes”) Surface of flow cell coated with a lawn of oligo pairs EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL Each piece has a unique sequence EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL “bridge PCR” EE Slawson Tempel, © WUSTL thousands of strands/cluster EE Slawson Tempel, © WUSTL each cluster (“polony”) has a unique sequence EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL EE Slawson Tempel, © WUSTL STOP STOP STOP STOP EE Slawson Tempel, © WUSTL Metzger M (2009) Nature Reviews Genetics 11: 31-46 STOP STOP STOP STOP EE Slawson Tempel, © WUSTL STOP STOP STOP OH EE Slawson Tempel, © WUSTL STOP STOP OH EE Slawson Tempel, © WUSTL STOP STOP EE Slawson Tempel, © WUSTL STOP EE Slawson Tempel, © WUSTL STOP EE Slawson Tempel, © WUSTL STOP EE Slawson Tempel, © WUSTL STOP OH EE Slawson Tempel, © WUSTL STOP STOP OH EE Slawson Tempel, © WUSTL STOP STOP OH EE Slawson Tempel, © WUSTL STOP STOP EE Slawson Tempel, © WUSTL STOP EE Slawson Tempel, © WUSTL STOP EE Slawson Tempel, © WUSTL STOP EE Slawson Tempel, © WUSTL STOP OH EE Slawson Tempel, © WUSTL G… © Illumina, EEST, © WUSTL GC… © Illumina, EEST, © WUSTL GCT… © Illumina, EEST, © WUSTL GCTG… © Illumina, EEST, © WUSTL GCTGA… © Illumina, EEST, © WUSTL 100+ Million Clusters Per Flow Cell 100 Microns Camera time is the limiting step! Flowcell 8 lanes For picture taking: Each lane is broken up into 100 tiles, each fluor is imaged separately – 2400 pictures taken per cycle EE Slawson Tempel, © WUSTL Chemistry problem 1: terminator is retained out of phase STOP EE Slawson Tempel, © WUSTL Chemistry problem 2: fluor is retained OH EE Slawson Tempel, © WUSTL Chemistry problem 2: fluor is retained STOP EE Slawson Tempel, © WUSTL Chemistry problem 2: fluor is retained STOP EE Slawson Tempel, © WUSTL Illumina >100 Gb/run HiSeq 90x106 reads/lane * 102 bp/read = 9x109 bp/lane * 16 lanes/run = 144 Gb/run ~ 3 – 30 Gb/run GAII Read length 30 – 120 bp ABI SOLiD Support Oligonucleotide Ligation Detection emPCR ABI SOLiD Mardis ER. (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet. 2008;9:387-402. ABI SOLiD ABI SOLID Mardis ER. (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet. 2008;9:387-402. ABI SOLiD Mardis ER. (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet. 2008;9:387-402. Mardis ER. (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet. 2008;9:387-402. Ion Torrent Nature 475:348 (2011) ~100 bp reads 30 Mb/run Ion Torrent read quality • 3.3 million SNPs 454, 7.4X, 24.5 Gb cost < $1M 10,654 cause aa substitution (7,648 different from Venter) • 222,718 indels (2 to 40kb) • 18 CNVs (26 kb to 1.6 Mb) • carrier of 10 highly penetrant disease alleles PMID: 20010809 Illumina, 73X, 173 Gb contig N50 = 40 kb scaffold N50 = 1.3 Mb PLoS Genetics 6: e1000832 (2010) ABI SOLiD, 30X coverage •107.5 Gb of raw data •55.51 Gb mapped to genome • 35 interchromosomal translocations • 1,315 structural variations (>100 bp) • 191,743 small (<21 bp) indels • 2,384,470 SNVs • 512 genes homozygously mutated GENETICS 2009 182: 25–32 a recessive EMS-induced mutation affecting egg shell morphology Illumina, 8X coverage • 103 SNP differences between mutant and wt • 9 non-synonomous • 2 nonsense >> one in encore, an obvious candidate 30 volume 42 | number 1 | january 2010 Illumina 5.1 Gb of sequence 76 bp reads 40X coverage 4 affected individuals RNA-Seq Pepke S, Wold B & Mortazavi A. (2009) Nature Methods 6:S22 ChIP-Seq Lefrançois P et al. (2009) Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing. BMC Genomics 10:37 Plant Physiology, July 2009, Vol. 150, pp. 1541–1555 rd 3 Generation (“Next2Gen”) Sequencing Technologies “Fabulous” having no basis in reality; mythical A T G C . . . . . . A T G C . . . . . . A T G C . .. .. . A T G C . . . . . . A T G . . . A T G C . . .. . . A T G C . . . Helicos Single-molecule sequencing Gupta PK. (2008) Single-molecule DNA sequencing technologies for future genomics research. Trends Biotechnol. 26:602-11 Metzger M (2009) Nature Reviews Genetics 11: 31-46 Helicos 105 to 140 Megabases per hour ~ 35 bp average read length (2009) Volume 27: 847 Helicos, 28X coverage, 84 Gb • 2.8M SNPs • 752 CNVs Ion Torrent Single-molecule sequencing Single-molecule sequencing + Gupta PK. (2008) Single-molecule DNA sequencing technologies for future genomics research. Trends Biotechnol. 26:602-11 Nanopore sequencing + - Gupta PK. (2008) Single-molecule DNA sequencing technologies for future genomics research. Trends Biotechnol. 26:602-11 Nanopore sequencing Gupta PK. (2008) Single-molecule DNA sequencing technologies for future genomics research. Trends Biotechnol. 26:602-11 Pacific Biosciences Single-molecule sequencing Eid et al 2008 emission excitation emission ZMW: a hole, tens of nanometers in diameter, fabricated in a 100nm metal film deposited on a silicon dioxide substrate detection volume 20 zeptoliters (10-21 liters). PacBio technology backgrounder: http://www.pacificbiosciences.com/index.php?q=technology-introduction PacBio technology backgrounder: http://www.pacificbiosciences.com/index.php?q=technology-introduction When the DNA polymerase encounters the nucleotide complementary to the next base in the template, it is incorporated into the growing DNA chain. During incorporation, the enzyme holds the nucleotide in the ZMWs detection volume for tens of milliseconds, orders of magnitude longer than the average diffusing nucleotide. The system detects this as a flash of bright light because the background is very low. The polymerase advances to the next base and the process continues to repeat PacBio technology backgrounder: http://www.pacificbiosciences.com/index.php?q=technology-introduction multiple reads of the same molecule PacBio technology backgrounder: http://www.pacificbiosciences.com/index.php?q=technology-introduction Eid J et al. (2009) Molecules Real-Time DNA Sequencing from Single Polymerase Molecules. Science 323, 133 PMID: 19023044 Does it work? • 150 bp circular template • ~93% raw accuracy • 15x coverage 99.3% accuracy Eid et al., 2009 ~ 2-5 bp/sec PacBio claims that, by 2013, the technology will be able to give a ‘raw’ human genome sequence in less than 3 min, and a complete high-quality sequence in 15 min. (http://www.bio-itworld.com/BioIT_Content.aspx?id=71746andamp;terms=Feb+12+2008+Pacific+Biosciences). Gupta PK. (2008) Single-molecule DNA sequencing technologies for future genomics research. Trends Biotechnol. 26:602-11 F. Sanger, S. Nicklen, and A. R. Coulson, Proc Natl Acad Sci U S A. 1977; 74: 5463–5467