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Synthesis & Analysis on Molecular Arrays

Synthesis & Analysis on Molecular Arrays. CHI Microarrays in Medicine 4-May-2005 9:20-9:50 AM. Thanks to: Washington U, Harvard-MIT Broad Inst., DARPA-BioSpice, DOE-GTL , EU-MolTools, NGHRI-CEGS, NHLBI-PGA, NIGMS-SysBio, PhRMA, Lipper Foundation

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Synthesis & Analysis on Molecular Arrays

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  1. Synthesis &Analysis on Molecular Arrays CHI Microarrays in Medicine 4-May-2005 9:20-9:50 AM 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

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

  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/Chip18 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 NimblegenPhotolabile 5'protection Nuwaysir, Smith, Albert Tian, Gong, Church

  5. Improve DNA Synthesis Cost Synthesis on chips in pools is 5000Xless 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) => ds-90-mer => ds-50-mer 20-mer PCR primers with restriction sites at the 50mer junctions Tian, Gong, Sheng , Zhou, Gulari, Gao, Church

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

  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) 50 75 125 225 425 825 … 100*2^(n-1) 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

  9. 0 1 2 3 4 PAM cycle# 5 50 75 125 225 425 #bp 825 Genome assembly in vitro PCR ] ] 50 HS PAM 425 MutS PAM 10K anneal 100K l-red5Mbp 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

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

  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. Western blot based on His-tags Tian & Church

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

  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 Projectdiagnosis, 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. Polymerase colony (polony) PCR in a gel B A’ A’ A’ B B B A’ B B B A’ A’ A’ A’ B A’ B B Single Molecule From Library A’ Primer is Extended by Polymerase A Primer A has 5’ immobilizing Acrydite 1st Round of PCR 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) Reduce or photo- cleave Mitra,RD, Shendure,J, Olejnik,J, Olejnik,EK, and Church,GM (2003) Fluorescent in situ Sequencing on Polymerase Colonies. Analyt. Biochem. 320:55-65

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

  19. Haplotypes inferred by pedigreevs. direct single molecule measures C A G C G C C A G C G C T T A T A T T T A T A T homozygous in the parents 1.8Mb 79.9Mb 88.2Mb 89.4Mb 114Mb 155Mb GM12248 GM12249 GM10835 rs3778973 rs997906 rs1557917 rs39284 rs10500042 rs4717028 C A G C G C T T A T A T heterozygous in the son

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

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

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

  23. Selector bead Plone-bead Fluorescent In SituSequencing in vitro Libraries 1 to 100kb Genomic 2x20bp after MmeI LR M M Sequencing primers PCR bead Greg Porreca Abraham Rosenbaum 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. Plone-bead FISSeq '04 '05 Consider amplification , homopolymer, context errors? • # of bases sequenced (total Mbp) 23 (no) 10.8 (yes) • # bases sequenced (unique) 73 b 4.7 Mb (72%) • Avg fold coverage 324K 2.3 • Pixels used per bead (analysis) 3.6 3.6 • Read Length (bp) 14 24 • Indels 0.6% ? • Substitutions (raw error-rate) 4e-51e-2 • Throughput (kb/min) 360 10 • Speed/cost ratio relative to 1100 32 current ABI capillary sequencing @ 0.75 kb/min/device 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. CD44 RNA splicing isoforms Eph4 = murine mammary epithelial cell line Eph4bDD = stable transfection of Eph4 with MEK-1 (tumorigenic) Zhu J, Shendure J, Mitra RD, Church GM. Science 301:836-8. Single molecule profiling of alternative pre-mRNA splicing.

  28. Soluble CD44 Zhu & Varma

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

  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. Synthesis &Analysis on Molecular Arrays CHI Microarrays in Medicine 4-May-2005 9:20-9:50 AM 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

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