Molecular Genomic Imaging Center (CEGS) Harvard / Wash U George Church, Rob Mitra Greg 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

Polymerase colony 2 vs. 1 immobilized primer in situ polonies vs. emulsion PCR beads single molecule vs. multi-molecule detection dNTP 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)

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

~1 kb genomic fragment paired genomic tags (17 to 18 bp each) common sequences MmeI Fisseq-F -R T30Tag 2Tag 1 Fisseq-FLeftRight Mid Seq2Seq1 In vitro construction of a complex, mate-paired library 43 bp Total = bp amplicon

(1)Emulsion PCR to 1 micron beads Dressman et al. PNAS'03 Template Amplification

Enrichment by Hybridization Selector Bead

One of 750 megapixel frames of gel-immobilized 1.0 micron beads, 0.3 micron pixels, 4-colors

ACUCAUC… (3’)…TAGAGT????????????????TGAGTAG…(5’) 5’-Cy5-nnnnAnnnn-3’ 5’-Cy3-nnnnGnnnn-3’ 5’-TR-nnnnCnnnn-3’ 5’-Cy3+Cy5-nnnnTnnnn-3’ 5'PO 4 Sequencing by Ligation (SBL) with fluorescent combinatorial 9-mers Excitation Emission nm

Consensus AccuracyFalse Positives (E.coli)False Positives (Human) 1E-34,0003,000,000 1E-4 BERMUDA/ABI ,000 1E-6Polony-SBL Goal of Resequencing  Discovery of Uncommon Variation Why low error rates?

 trp/  tyrA pair of genomes shows the best co-growth (syntrophs) Reppas & Lin First Passage SecondPassage Genome engineering: Select for cross-feeding

Co-evolution of cross-feeding Trp- & Tyr- genome pair

~1 kb genomic fragment 980 ± 96 bp ~860,000 independent mate-pairing events

confirmed 776 bp deletion via tandem 8 bp repeats 1,974,001 (MG1655) 1,978,000 (MG1655) Aberrations in mate-pair distance indicative of rearrangements

Base-calling Tetrahedron Fluorescent SBL data quality measured by distance to the 4 vertices. A G C T

Mean accuracy = 99.5% Best 50% of base-calls are 99.9% accurate Q40 Q30 Q20 Raw Error Rate

Consensus error rates

PositionTypeGeneLocation ABI Confirmation Comments 986,334T > GompFTATA box Only in evolved strain 931,9608 bp dellrpframeshift Only in evolved strain 1,976, bp delinsB_5IS element MG1655 heterogeneity 3,957,960C > TppiC5' UTR MG1655 heterogeneity 4,654,533T > CcIGlu > Glu heterogeneity 4,647,960T > CORF61Lys > Gly heterogeneity 985,797T > GompFGlu > Ala(in progress) 454,864T > CtigGly > Gly (in progress) 4,648,691G > AexoPhe > Phe (in progress) Mutation Discovery in Engineered & Evolved Trp - Strain

ABI2004 Jun >2007 # bp/expt-2e73e7 3e8 60e9 Complexity (bp) -744e6 3e9 6e9 Avg Fold Cov83e Pix per bp Read-length (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-30.6%1e-3 1e-3 1e-3 Subst Error 4e-34e-61e-3 1e-3 1e-3 3X Cons Err 1e-4 - 1e-6 3e-7 1e-7 Kb / min e3 1e6 Pix / sec-2e52e6 6e6 2e7 Enz $/mg Cost comparison & projection

>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

Assume paired 17-mers (i.e. read full tag length) with bp distance distribution (980 ±  = 96 bp observed) Exact Matching (34/34) Zero UniqueMultiple Paired, no substitutions Paired, one substitution Unpaired, no substitutions Single Substitution or Exact (33/34 or 34/34) ZeroUniqueMultiple Paired, no substitutions Paired, one substitution Unpaired, no substitutions Human Resequencing with Mate-Paired 17 bp Tags [simulation]

rs rs rs39284 rs rs GM10835 CGCGCCGCGC TATATTATAT TT=137 CT=2 (TC=1) CC= Mb Single chromosome molecule haplotypes

Amplifying & sequencing whole genomes from single cells  29 real-time amplification No template control Affymetrix quantitation of 2 independent amplifications Escherchia & Prochlorococcus Zhang, Martiny, Chisholm, Church, unpub.

Polymerase colony 2 vs. 1 immobilized primer in situ polonies vs. emulsion PCR beads single molecule vs. multi-molecule detection dNTP 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)

Shared Resources STTR Polymerase libraries NEB MJR ABI Fuller CCDs spectra, cost, #pixels, sensistivity, speed software Cancer Genome NCAB clonal? enrichment MRD accuracy read length Cost estimates distribute template spreadsheet Roundtable I