1. 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

2. 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)

3. 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

4. ~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 32 25 Total = 134-136 bp amplicon

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

6. Enrichment by Hybridization Selector Bead

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

8. 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 647 700 555 605 572 630 555 700 nm

9. Consensus AccuracyFalse Positives (E.coli)False Positives (Human) 1E-34,0003,000,000 1E-4 BERMUDA/ABI 400 300,000 1E-6Polony-SBL 4 3000 Goal of Resequencing  Discovery of Uncommon Variation Why low error rates?

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

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

12. ~1 kb genomic fragment 980 ± 96 bp ~860,000 independent mate-pairing events

13. 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

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

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

16. Consensus error rates

17. PositionTypeGeneLocation ABI Confirmation Comments 986,334T > GompFTATA box Only in evolved strain 931,9608 bp dellrpframeshift Only in evolved strain 1,976,500776 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

18. ABI2004 Jun 2005 2006 >2007 # bp/expt-2e73e7 3e8 60e9 Complexity (bp) -744e6 3e9 6e9 Avg Fold Cov83e5 60.1 10 Pix per bp-300 1724333 1 Read-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-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 0.8360 27 1e3 1e6 Pix / sec-2e52e6 6e6 2e7 Enz $/mg-888 0.4 Cost comparison & projection

19. >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

20. 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 UniqueMultiple Paired, no substitutions ----94.45.6 Paired, one substitution 98.30.51.3 Unpaired, no substitutions 98.80.30.9 Single Substitution or Exact (33/34 or 34/34) ZeroUniqueMultiple Paired, no substitutions ----90.49.7 Paired, one substitution ----92.87.2 Unpaired, no substitutions 96.01.52.5 Human Resequencing with Mate-Paired 17 bp Tags [simulation]

21. rs3778973 rs1557917 rs39284 rs10500042 rs4717028 GM10835 CGCGCCGCGC TATATTATAT TT=137 CT=2 (TC=1) CC=131 153Mb Single chromosome molecule haplotypes

22. 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.

23. 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)

24. 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