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2 nd Generation (NextGen) Sequencing Technologies Fantastic bizarre or exotic; seeming more appropriate to a fairy tale than to reality or practical use

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Read Length is Not As Important For Resequencing Jay Shendure

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Paired End Reads are Important! Repetitive DNA Unique DNA Single read maps to multiple positions Paired read maps uniquely Read 1Read 2 Known Distance

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emulsion PCR Margulies M et al., (2006) Genome sequencing in microfabricated high-density picolitre reactors Nature 437, 376-380 emPCR

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Roche 454 Margulies M et al., (2006) Genome sequencing in microfabricated high-density picolitre reactors Nature 437, 376-380

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OH EE Slawson Tempel, WUSTL

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OH EE Slawson Tempel, WUSTL

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OH EE Slawson Tempel, WUSTL

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OH EE Slawson Tempel, WUSTL

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OH 3 2 1 EE Slawson Tempel, WUSTL

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OH Pyrophosphate EE Slawson Tempel, WUSTL

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OH EE Slawson Tempel, WUSTL

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OH ATP + luciferin EE Slawson Tempel, WUSTL

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OH ATP + luciferin EE Slawson Tempel, WUSTL

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OH EE Slawson Tempel, WUSTL OH

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EE Slawson Tempel, WUSTL OH

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EE Slawson Tempel, WUSTL

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OH EE Slawson Tempel, WUSTL

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OH EE Slawson Tempel, WUSTL

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OH EE Slawson Tempel, WUSTL

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OH EE Slawson Tempel, WUSTL

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OH EE Slawson Tempel, WUSTL

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OH EE Slawson Tempel, WUSTL

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OH ATP + luciferin EE Slawson Tempel, WUSTL

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OH ATP + luciferin EE Slawson Tempel, WUSTL

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1- mer 2-mer 3-mer 4-mer Brightness of flash is proportional to number of nucleotides added Flash brightness TCACTTCAAGGGT Flash is too bright EE Slawson Tempel, WUSTL

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A T G C Read length 350-400 bp 200 cycles EE Slawson Tempel, WUSTL ~ 0.5 Gb/run Roche 454

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EE Slawson Tempel, WUSTL Nebulizer ~ 400 bp Illumina

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EE Slawson Tempel, WUSTL

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Flow cell 8 channels (lanes) Surface of flow cell coated with a lawn of oligo pairs

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EE Slawson Tempel, WUSTL

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Each piece has a unique sequence EE Slawson Tempel, WUSTL

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bridge PCR

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thousands of strands/cluster each cluster (polony) has a unique sequence EE Slawson Tempel, WUSTL

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STOP EE Slawson Tempel, WUSTL

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Metzger M (2009) Nature Reviews Genetics 11: 31-46

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STOP EE Slawson Tempel, WUSTL

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OH STOP EE Slawson Tempel, WUSTL

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OH STOP EE Slawson Tempel, WUSTL

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STOP EE Slawson Tempel, WUSTL

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STOP EE Slawson Tempel, WUSTL

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STOP EE Slawson Tempel, WUSTL

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STOP EE Slawson Tempel, WUSTL

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STOP OH EE Slawson Tempel, WUSTL

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OH STOP EE Slawson Tempel, WUSTL

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OH STOP EE Slawson Tempel, WUSTL

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STOP EE Slawson Tempel, WUSTL

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STOP EE Slawson Tempel, WUSTL

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STOP EE Slawson Tempel, WUSTL

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STOP EE Slawson Tempel, WUSTL

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STOP OH EE Slawson Tempel, WUSTL

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GG Illumina, EEST, WUSTL

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GCGC

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GCTGCT

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GCTGGCTG

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GCTGAGCTGA

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100+ Million Clusters Per Flow Cell 100 Microns

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Flowcell 8 lanes For picture taking: Each lane is broken up into 100 tiles, each fluor is imaged separately 2400 pictures taken per cycle Camera time is the limiting step! EE Slawson Tempel, WUSTL

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STOP Chemistry problem 1: terminator is retained EE Slawson Tempel, WUSTL out of phase

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OH Chemistry problem 2: fluor is retained EE Slawson Tempel, WUSTL

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STOP EE Slawson Tempel, WUSTL Chemistry problem 2: fluor is retained

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STOP EE Slawson Tempel, WUSTL Chemistry problem 2: fluor is retained

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Read length 30 120 bp ~ 3 30 Gb/run GAII Illumina >100 Gb/run HiSeq 90x10 6 reads/lane * 10 2 bp/read = 9x10 9 bp/lane * 16 lanes/run = 144 Gb/run

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ABI SOLiD emPCR Support Oligonucleotide Ligation Detection

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ABI SOLiD Mardis ER. (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet. 2008;9:387-402.

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ABI SOLiD Mardis ER. (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet. 2008;9:387-402.

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ABI SOLID Mardis ER. (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet. 2008;9:387-402.

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ABI SOLiD Mardis ER. (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet. 2008;9:387-402.

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Ion Torrent

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Nature 475:348 (2011) ~100 bp reads 30 Mb/run

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Ion Torrent read quality

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454, 7.4X, 24.5 Gb cost < $1M 3.3 million SNPs 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

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PMID: 20010809 Illumina, 73X, 173 Gb contig N50 = 40 kb scaffold N50 = 1.3 Mb

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35 interchromosomal translocations 1,315 structural variations (>100 bp) 191,743 small (

Illumina, 8X coverage 103 SNP differences between mutant and wt 9 non-synonomous 2 nonsense >> one in encore, an obvious candidate GENETICS 2009 182: 2532 a recessive EMS-induced mutation affecting egg shell morphology

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30 volume 42 | number 1 | january 2010 Illumina 5.1 Gb of sequence 76 bp reads 40X coverage 4 affected individuals

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Pepke S, Wold B & Mortazavi A. (2009) Nature Methods 6:S22 RNA-Seq

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ChIP-Seq Lefranois P et al. (2009) Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing. BMC Genomics 10:37

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Plant Physiology, July 2009, Vol. 150, pp. 15411555

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3 rd Generation (Next 2 Gen) Sequencing Technologies Fabulous having no basis in reality; mythical

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...ATGC......ATGC......ATGC......ATGC......ATGC......ATGC......ATGC......ATGC......ATGC......ATGC......ATGC......ATGC......ATGC......ATGC...

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Helicos Single-molecule sequencing

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Gupta PK. (2008) Single-molecule DNA sequencing technologies for future genomics research. Trends Biotechnol. 26:602-11

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Metzger M (2009) Nature Reviews Genetics 11: 31-46

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105 to 140 Megabases per hour ~ 35 bp average read length Helicos

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(2009) Volume 27: 847 Helicos, 28X coverage, 84 Gb 752 CNVs 2.8M SNPs

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Ion Torrent Single-molecule sequencing

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Gupta PK. (2008) Single-molecule DNA sequencing technologies for future genomics research. Trends Biotechnol. 26:602-11 + - Single-molecule sequencing

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Nanopore sequencing Gupta PK. (2008) Single-molecule DNA sequencing technologies for future genomics research. Trends Biotechnol. 26:602-11 + -

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Nanopore sequencing Gupta PK. (2008) Single-molecule DNA sequencing technologies for future genomics research. Trends Biotechnol. 26:602-11

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Eid et al 2008 Pacific Biosciences Single-molecule sequencing

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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 excitationemission

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PacBio technology backgrounder: http://www.pacificbiosciences.com/index.php?q=technology-introduction

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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

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multiple reads of the same molecule PacBio technology backgrounder: http://www.pacificbiosciences.com/index.php?q=technology-introduction

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Eid J et al. (2009) Molecules Real-Time DNA Sequencing from Single Polymerase Molecules. Science 323, 133 PMID: 19023044

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Does it work? 150 bp circular template ~93% raw accuracy 15x coverage 99.3% accuracy Eid et al., 2009

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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 ~ 2-5 bp/sec

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F. Sanger, S. Nicklen, and A. R. Coulson, Proc Natl Acad Sci U S A. 1977; 74: 54635467