molecular genomic imaging center (cegs) harvard / wash u george church, rob mitra greg porreca, jay...
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![Page 1: Molecular Genomic Imaging Center (CEGS) Harvard / Wash U George Church, Rob Mitra Greg Porreca, Jay Shendure Sequencing by Ligation on Polony Beads with](https://reader036.vdocument.in/reader036/viewer/2022062322/56649d435503460f94a1e7c6/html5/thumbnails/1.jpg)
Molecular Genomic Imaging Center (CEGS)Harvard / Wash U
George Church, Rob MitraGreg Porreca, Jay Shendure
Sequencing by Ligation on Polony Beads
with Nick Reppas, Kun Zhang, Shawn Douglas, Mike Wang,
Abraham Rosenbaum, Agencourt
Personal Genomics, Stem Cells, ELSI
Synthetic Biology
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Polymerase colony
2 vs. 1 immobilized primerin situ polonies vs. emulsion PCR beadssingle molecule vs. multi-molecule detectiondNTP extension (SBE) vs. ligation (SBL) (>=3X error 1e-6, 1/10 cost of ABI E.coli )
Shendure, Porreca, Mitra, Church
• Single chromosomes : haplotyping (Zhang)• Single cells : full sequence (Zhang & Martiny)• Single RNA molecules : RNA splicing (Zhu, Varma)
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1. In vitro construction of a complex mate-paired library
2. Template amplification to one micron beads by emulsion PCR
3. Cyclic Array Sequencing by Ligation (SBL)
Polony Sequencing Overview
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~1 kb genomic fragment
paired genomic tags
(17 to 18 bp each)
common sequences
MmeI
Fisseq-F Fisseq-RT30 Tag 2Tag 1Fisseq-FLeft RightMid Seq2Seq1
In vitro construction of a complex, mate-paired library
43 bp 32 25
Total = 134-136 bp amplicon
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(1) Emulsion PCR
to 1 micron beads
Dressman et al. PNAS'03
Template Amplification
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Enrichment by Hybridization
Selector Bead
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One of 750 megapixel frames of gel-immobilized 1.0 micron beads, 0.3 micron pixels, 4-colors
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ACUCAUC…(3’)…TAGAGT????????????????TGAGTAG…(5’)
5’-Cy5-nnnnAnnnn-3’ 5’-Cy3-nnnnGnnnn-3’ 5’-TR-nnnnCnnnn-3’ 5’-Cy3+Cy5-nnnnTnnnn-3’
5'PO4
Sequencing by Ligation (SBL) with fluorescent combinatorial 9-mers
Excitation Emission 647 700 555 605 572 630 555 700
nm
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Consensus Accuracy False Positives (E.coli) False Positives (Human)1E-3 4,000 3,000,000
1E-4 BERMUDA/ABI 400 300,000
1E-6 Polony-SBL 4 3000
Goal of Resequencing Discovery of Uncommon Variation
Why low error rates?
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trp/tyrA pair of genomes shows the best co-growth
(syntrophs) Reppas & Lin
First Passage SecondPassage
Genome engineering:Select for cross-feeding
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0
1
2
3
4
5
6
7
8
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
# of passages
Do
ub
lin
g t
ime
(h
r)
Q1
Q3
Q2-1
Q2-2
EcNR1
Co-evolution of cross-feedingTrp- & Tyr- genome pair
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~1 kb genomic fragment
980 ± 96 bp
~860,000 independent mate-pairing events
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0 1000 2000 3000 4000 5000 6000 70000
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
confirmed 776 bp deletionvia tandem 8 bp repeats
1,974,001(MG1655)
1,978,000(MG1655)
Aberrations in mate-pair distance indicative of rearrangements
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Base-calling Tetrahedron
Fluorescent SBL data quality measured by distance to the 4 vertices.
A
G
C
T
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Mean accuracy = 99.5%
Best 50% of base-calls are 99.9% accurate
1E-04
1E-03
1E-02
Cumulative Fraction of Data
Q40
Q30
Q20
Raw Error Rate
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Mutation
Coverage % genome Error Mock SNPs Candidates
>=3x 68.10% < 1e- 6 60/ 60 correct n = 7
2x 13.40% 0.0002 15/ 15 correct n = 108
1x 11.20% 0.007 11/ 14 correct n = 3344
1E-71E-61E-51E-41E-31E-21E-1
Coverage
Consensus error rates
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Position Type Gene LocationABI
Confirmation
Comments
986,334 T > G ompF TATA box Only in evolved strain
931,960 8 bp del lrp frameshift Only in evolved strain
1,976,500 776 bp del insB_5 IS element MG1655 heterogeneity
3,957,960 C > T ppiC 5' UTR MG1655 heterogeneity
4,654,533 T > C cI Glu > Glu heterogeneity
4,647,960 T > C ORF61 Lys > Gly heterogeneity
985,797 T > G ompF Glu > Ala (in progress)
454,864 T > C tig Gly > Gly(in progress)
4,648,691 G > A exo Phe > Phe(in progress)
Mutation Discovery in Engineered & Evolved Trp-Strain
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ABI 2004 Jun 2005 2006 >2007
# bp/expt - 2e7 3e7 3e8 60e9
Complexity (bp) - 74 4e6 3e9 6e9
Avg Fold Cov 8 3e5 6 0.1 10
Pix per bp - 300 1724 333 1Read-length 900 14 (SBE) 25 (pair) 35 42
$ / Q20 kb 8e-1 - 8e-2 4e-2 1e-5
$/ 1X 3e9 b 2e6 - 2e5 5e4 1e2 (2e3)
Indel Error 5e-3 0.6% 1e-3 1e-3 1e-3
Subst Error 4e-3 4e-6 1e-3 1e-3 1e-3
3X Cons Err 1e-4 - 1e-6 3e-7 1e-7
Kb / min 0.8 360 27 1e3 1e6
Pix / sec - 2e5 2e6 6e6 2e7Enz $/mg - 8 8 8 0.4
Cost comparison & projection
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>2007
# bp/expt 60e9 20X of 3e9 = 10X diploid
Complexity (bp) 6e9 Automated 96-well libraries
Avg Fold Cov 10 (Currently align .4 pix = .1 micron)
Pix per bp 1 Sensitivity & align CCD & slide?
Read-length 42 Is 34 enough? (next slide)
$ / Q20 kb 1e-5 (20X 3e9)
$/ 1X 3e9 b 1e2 (2e3) Need haplotyping too? (slide after next)
Indel Error 1e-3
Subst Error 1e-3
3X Cons Err 1e-7
Kb / min 1e6
Pix / sec 2e7 Current camera is 3e7, but stage is 2e6
Enz $/mg 0.4 Realized for many recombinant proteins
Challenges in $2000 genome
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Assume paired 17-mers (i.e. read full tag length) with 750-1150 bp distance distribution (980 ± =96 bp observed)
Exact Matching (34/34) Zero UniqueZero Unique MultipleMultiple
Paired, no substitutions ---- 94.4 5.6Paired, one substitution 98.3 0.5 1.3Unpaired, no substitutions 98.8 0.3 0.9
Single Substitution or Exact (33/34 or 34/34) Zero Unique Multiple
Paired, no substitutions ---- 90.4 9.7Paired, one substitution ---- 92.8 7.2Unpaired, no substitutions 96.0 1.5 2.5
Human Resequencing with Mate-Paired 17 bp Tags [simulation]
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rs3778973
rs1557917
rs39284
rs10500042
rs4717028
GM10835
C
G
C
G
C
T
A
T
A
T
TT=137 CT=2 (TC=1) CC=131
153Mb
Single chromosome molecule haplotypes
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Amplifying & sequencing whole genomes from
single cells
29 real-time amplification
No template control
Affymetrix quantitation of 2 independent amplifications
Escherchia & ProchlorococcusZhang, Martiny,
Chisholm, Church, unpub.
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Polymerase colony
2 vs. 1 immobilized primerin situ polonies vs. emulsion PCR beadssingle molecule vs. multi-molecule detectiondNTP extension (SBE) vs. ligation (SBL) (>=3X error 1e-6, 1/10 cost of ABI E.coli )
Shendure, Porreca, Mitra, Church
• Single chromosomes : haplotyping (Zhang)• Single cells : full sequence (Zhang & Martiny)• Single RNA molecules : RNA splicing (Zhu, Varma)
![Page 24: Molecular Genomic Imaging Center (CEGS) Harvard / Wash U George Church, Rob Mitra Greg Porreca, Jay Shendure Sequencing by Ligation on Polony Beads with](https://reader036.vdocument.in/reader036/viewer/2022062322/56649d435503460f94a1e7c6/html5/thumbnails/24.jpg)
Shared Resources STTR Polymerase libraries NEB MJR ABI Fuller CCDs spectra, cost, #pixels, sensistivity, speed software
Cancer Genome 12500 NCAB clonal? enrichment MRD accuracy read length
Cost estimates distribute template spreadsheet
Roundtable I