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
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
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
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
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