1. Thanks to: Washington U, Harvard-MIT Broad Inst., DARPA-BioSpice, DOE-GTL, EU-MolTools, NGHRI-CEGS, NHLBI-PGA, NIGMS-SysBio, PhRMA, Lipper Foundation Agencourt, Ambergen, Atactic, BeyondGenomics, Caliper, Genomatica, Genovoxx, Helicos, MJR, NEN, Nimblegen, SynBioCorp, ThermoFinnigan, Xeotron/Invitrogen For more info see: arep.med.harvard.edu CHI Microarrays in Medicine 4-May-2005 9:20-9:50 AM Synthesis &Analysis on Molecular Arrays

2. Systems Biology Loop Syntheses & Perturbations Models Experimental designs (Systematic) Data Analysis & Synthesis Tools Genome engineering DNA & RNA Polony Sequencing

3. Why Synthetic Genomes & Proteomes? Test array hypotheses e.g. cis-DNA/RNA-elements Multi-epitopes, vaccines, protein design Mass spectrometry & array standards. Access to any protein (complex) including post-transcriptional modifications Utility of molecular biology DNA-RNA-Protein in vitro "kits" (e.g. PCR, T7, Roche) Whole genome or part? Whole if major redesign e.g. changing the genetic code and stability.

4. Up to 760K Oligos/Chip 18 Mbp for $1K raw (6-18K genes) <1K Oxamer Electrolytic acid/base 8K Atactic/Xeotron/Invitrogen Photo-Generated Acid Sheng, Zhou, Gulari, Gao (U.Houston) 24K Agilent Ink-jet standard reagents 48K Febit 100K Metrigen 380K Nimblegen Photolabile 5'protection Nuwaysir, Smith, Albert Tian, Gong, Church

5. Improve DNA Synthesis Cost Synthesis on chips in pools is 5000X less expensive per oligonucleotide, but amounts are low (1e6 molecules rather than usual 1e12) & bimolecular kinetics slow with square of concentration decrease!) Solution: Amplify the oligos then release them. 10 50 10 => ss-70-mer (chip) 20-mer PCR primers with restriction sites at the 50mer junctions Tian, Gong, Sheng, Zhou, Gulari, Gao, Church => ds-90-mer => ds-50-mer

6. Improve DNA Synthesis Accuracy via mismatch selection Tian & Church Other mismatch methods: MutS (&H,L)

7. Improving DNA synthesis accuracy Method Bp/error Chip assembly only 160 Hybridization-selection 1,400 MutS-gel-shift 10,000 PCR 35 cycles 10,000 MutHLS cleavage 100,000 Tian & Church 2004 Nature Carr & Jacobson 2004 NAR Smith & Modrich 1997 PNAS http://www.invitrogen.com/content.cfm?pageid=453

8. Computer aided Design Polymerase Assembly Multiplexing (CAD-PAM) For tandem, inverted and dispersed repeats: Focus on 3' ends, hierarchical assembly, size-selection and scaffolding. Mullis 1986 CSHSQB, Dillon 1990 BioTech, Stemmer 1995 Gene Tian et al. 2004 Nature, Kodumal et 2004 PNAS 50 75 125 225 425 825 … 100*2^(n-1)

9. Genome assembly 0 1 2 3 4 PAM cycle# 5 50 75 125 225 425 #bp 825 50 HS PAM 425 MutS PAM 10K anneal 100K  red  5Mbp USER USER-S1 USER-5'only One pool 480 pools 480 genomic 48 1 of 117K universal primers primer pairs HS=Hybridization-Selection USER=Uracil DNA glycosylase &EndoVIII remove flanking primer pairs ] ] PCR in vitro

10. All 30S-Ribosomal-protein DNAs (codon re-optimized) Tian, Gong, Sheng, Zhou, Gulari, Gao, Church 1.7 kb 0.3 kb s19 0.3kb Nimblegen 95K chip Atactic <4K chip

11. Extreme mRNA makeover for protein expression in vitro RS-2,4,5,6,9,10,12,13,15,16,17,and 21 detectable initially. RS-1, 3, 7, 8, 11, 14, 18, 19, 20 initially weak or undetectable. Solution: Iteratively resynthesize all mRNAs with less mRNA structure. Tian & Church Western blot based on His-tags

12. 3 Exponential technologies Shendure J, Mitra R, Varma C, Church GM, 2004 Nature Reviews of Genetics. Carlson 2003 ; Kurzweil 2002 urea E.coli B12 tRNA operons telegraph Computation & communication (bits/sec) Synthesis (daltons) Analysis (bp/$) tRNA

13. Why Personal Genomics? Pathogen rapid response: emerging disease & biowarfare B & T-cell diversity: clinical temporal profiling Proteomics: antibodies & aptamers RNA & methylation: quantitate splicing, & chromatin. Preventative medicine: genotype–phenotype association Cancer: drug targets, loss-of-heterozygosity Synthetic biology : laboratory selections Phylogenetic: footprinting, biodiversity Shendure et al. 2004 Nature Reviews of Genetics

14. Cancer Genome Project diagnosis, prognosis, therapies Mutations G719S, L858R, Del746ELREA in red. EGFR Mutations in lung cancer: correlation with clinical response to gefitinib [Iressa] therapy. Paez, … Meyerson (Apr 2004) Science 304: 1497 Lynch … Haber, (Apr 2004) New Engl J Med. 350:2129. Pao.. Mardis,Wilson,Varmus H, PNAS (Aug 2004) 101:13306-11. Dulbecco R. (1986) A turning point in cancer research: sequencing the human genome. Science 231:1055-6.

15. A’ B B B B B B A Single Molecule From Library B B A’ 1st Round of PCR Primer is Extended by Polymerase B A’ B Polymerase colony (polony) PCR in a gel Primer A has 5’ immobilizing Acrydite Mitra & Church Nucleic Acids Res. 27: e34

16. Polymerase clones Plone sequencing Polony-slides vs. Plone-beads 1 vs. 2 immobilized primers dNTP extension vs. ligation Single molecule vs. multi-molecule detection

17. Cleavable dNTP-Fluorophore (& terminators) Mitra,RD, Shendure,J, Olejnik,J, Olejnik,EK, and Church,GM (2003) Fluorescent in situ Sequencing on Polymerase Colonies. Analyt. Biochem. 320:55-65 Reduce or photo- cleave

18. Polony Bead Sequencing Pipeline In vitro libraries via paired tag manipulation Bead polonies via emulsion PCR [Dre03] Monolayered immobilization in acrylamide Enrichment of amplified beads SOFTWARE Images → Tag Sequences Tag Sequences → Genome FISSEQ or “wobble” sequencing Epifluorescence Scope with Integrated Flow Cell Mitra, Shendure, Porreca, Rosenbaum, Church unpub.

19. rs3778973 rs997906 rs1557917 rs39284 rs10500042 rs4717028 CAGCGCCAGCGC CAGCGCCAGCGC GM12248GM12249 GM10835 TTATATTTATAT TTATATTTATAT CAGCGCCAGCGC TTATATTTATAT Haplotypes inferred by pedigree vs. direct single molecule measures homozygous in the parents heterozygous in the son 1.8Mb 79.9Mb 88.2Mb 89.4Mb 114Mb 155Mb

20. rs3778973 rs997906 rs1557917 rs39284 rs10500042 rs4717028 GM10835 1.8Mb 79.9Mb 88.2Mb 89.4Mb 114Mb 155Mb CAGCGCCAGCGC TTATATTTATAT 1Mb haplotypes AT=198 GT=0 GC=45

21. rs3778973 rs997906 rs1557917 rs39284 rs10500042 rs4717028 GM10835 1.8Mb 79.9Mb 88.2Mb 89.4Mb 114Mb 155Mb CAGCGCCAGCGC TTATATTTATAT 75Mb haplotypes TT=8 TC=0 AC=23

22. rs3778973 rs997906 rs1557917 rs39284 rs10500042 rs4717028 GM10835 1.8Mb 79.9Mb 88.2Mb 89.4Mb 114Mb 155Mb CAGCGCCAGCGC TTATATTTATAT 153Mb haplotypes TT=72 CT=15 CC=28

23. Plone-bead Fluorescent In Situ Sequencing in vitro Libraries Greg Porreca Abraham Rosenbaum 1 to 100kb Genomic M L R M PCR bead Sequencing primers Selector bead 2x20bp after MmeI Dressman et al PNAS 2003 emulsion

24. Plone- FISSeq : up to 1 billion beads/slide White= Fe-core pixels, Cy5 primer (570nm) ; Cy3 dNTP (666nm) Jay Shendure, Greg Porreca

25. # of bases sequenced (total Mbp)23 (no) 10.8 (yes) # bases sequenced (unique)73 b 4.7 Mb (72%) Avg fold coverage324K 2.3 Pixels used per bead (analysis) 3.6 3.6 Read Length (bp) 14 24 Indels 0.6% ? Substitutions (raw error-rate) 4e-5 1e-2 Throughput (kb/min) 360 10 Speed/cost ratio relative to 1100 32 current ABI capillary sequencing @ 0.75 kb/min/device Plone-bead FISSeq '04 '05 Consider amplification, homopolymer, context errors? Shendure & Porreca

26. CD44 Exon Combinatorics (Zhu & Shendure) Alternatively Spliced Cell Adhesion Molecule Specific variable exons are up-or-down-regulated in various cancers (>2000 papers) v6 & v7 enable direct binding to chondroitin sulfate, heparin… Zhu,J, et al. Science. 301:836-8.

27. Zhu J, Shendure J, Mitra RD, Church GM. Science 301:836-8. Single molecule profiling of alternative pre-mRNA splicing. Eph4 = murine mammary epithelial cell line Eph4bDD = stable transfection of Eph4 with MEK-1 (tumorigenic) CD44 RNA splicing isoforms

28. Soluble CD44 Zhu & Varma

29. Systems Biology Loop Syntheses & Perturbations Models Experimental designs (Systematic) Data Analysis & Synthesis Tools Genome engineering DNA & RNA Polony Sequencing

30. Molecular Systems Biology Transcriptomics Proteomics Metabolomics Functional genomics Structural genomics Computational biology Theoretical biology Mathematical biology Synthetic biology An open access journal www.nature.com/msb/

31. Thanks to: Washington U, Harvard-MIT Broad Inst., DARPA-BioSpice, DOE-GTL, EU-MolTools, NGHRI-CEGS, NHLBI-PGA, NIGMS-SysBio, PhRMA, Lipper Foundation Agencourt, Ambergen, Atactic, BeyondGenomics, Caliper, Genomatica, Genovoxx, Helicos, MJR, NEN, Nimblegen, SynBioCorp, ThermoFinnigan, Xeotron/Invitrogen For more info see: arep.med.harvard.edu CHI Microarrays in Medicine 4-May-2005 9:20-9:50 AM Synthesis &Analysis on Molecular Arrays

32. .