Personal Genomes & Medicine

1. Personal Genomes & Medicine HST Advisory Council Thursday 16-Nov-2004 2:00 to 2:20 PM Thanks to: Broad Inst.,DARPA-BioComp, DOE-GTL,EU-MolTools, NGHRI-CEGS, NHLBI-PGA, NIGMS-CECBSR,PhRMA, Lipper Foundation Agencourt, Ambergen, Atactic, BeyondGenomics, Caliper, Genomatica, Genovoxx, Helicos, MJR, NEN, Nimblegen, ThermoFinnigan, Xeotron/Invitrogen For more info see: arep.med.harvard.edu

2. Why sequence? • • Cancer: mutation sets for individual clones, loss-of-heterozygosity • • Pathogen "weather map", biowarfare sensors • • RNA splicing & chromatin modification patterns. • Synthetic biology & lab selections • Antibodies or "aptamers" for any protein • B & T-cell receptor diversity: Temporal profiling, clinical • Preventative medicine & genotype–phenotype associations • Cell-lineage during development • Phylogenetic footprinting, biodiversity Shendure et al. 2004 Nature Rev Gen 5, 335.

3. The idea of Common SNPs for Common Diseases has been hugely oversold. Do association studies need the added baggage of "linkage" assumptions? Should we determine genotype (haplotype) directly (at low cost) rather than infer it from population trends?

4. Rare Alleles / Common Diseases Even "dispensable" regions of the genome can harbor neomorphic alleles. Each of us has about 104 mutations since the last major population bottleneck. "-463GA, has been associated with incidence or severity of inflammatory diseases, including atherosclerosis and Alzheimer's disease, and some cancers. The polymorphism is within an Alu element " Kumar AP, et al. (2004) J Biol Chem. 279:8300-15. Variable breakpoints in Burkitt lymphoma cells with chromosomal t(8;14) translocation separate c-myc and the IgH locus up to several hundred kb. Joos S, et al. (1992) Hum Mol Genet. 1:625-32. Multiple rare alleles contribute to low plasma levels of HDL cholesterol. Cohen JC et al. (2004) Science. 305:869-72.

5. Personal genomics & cancer therapy Mutations G719S, L858R, Del746ELREA in red. EGFR Mutations in lung cancer: correlation with clinical response to gefitinib [Iressa] therapy. Paez, … Meyerson (2004) Science 304: 1497 Dulbecco R. (1986) A turning point in cancer research: sequencing the human genome. Science 231:1055-6.

6. Why 'single molecule' sequencing? (1) Single-cells: Preimplantation (PGD), uncultivatable (2) Co-occurrence on a molecule, complex, cell RNA splice-forms & DNA haplotypes (3) Cost: $1K-100K "personal genomes" http://grants.nih.gov/grants/guide/rfa-files/RFA-HG-04-003.html (4) Precision: Counting 109 RNA tags (to reduce variance) (~5e5RNAs per human cell) Fixed 5e3 5e4 5e6 5e9 (goal) costs EST SAGE MPSS Polony-FISSeq (polymerase colony)

7. 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.

8. CD44 RNA 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.

9. Multi-locus haplotyping on pooled samples Throughput = (# loci × # samples) / time Kun Zhang

10. Multi-locus haplotyping NOS3 Chr 7 C/T G/A G/T G/A T/A C/T C/T ~24-Kb

11. IL6-3572 : A/C ~60-Mb CD36-4366 : T/A Chromosome-wide haplotyping Human Chr. 7 A..A A..T

12. Convergence on non-electrophorectic tag-sequencing methods? • Tag >400 14-26 20 100 26 bp (2-ends) • EST SAGE MPSS 454 Polony-Seq • Ronaghi • Single-molecule vs. amplified single molecule. • Array vs. bead packing vs. random • Rapid scans vs. long scans (chemically limited, 454) • Number of immobilized primers: • 0: Chetverin'97 "Molecular Colonies" • 1: Mitra'99 > Agencourt "Bead Polonies" • 2: Kawashima'88, Adams'97 > Lynx/Solexa: "Clusters" http://arep.med.harvard.edu/Polonator/Plone.htm

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

14. Selector bead Polony Fluorescent In SituSequencing Libraries 1 to 100kb Genomic 2x20bp after MmeI (BceAI, AcuI) LR M M Sequencing primers PCR bead Greg Porreca Abraham Rosenbaum Dressman et al PNAS 2003 emulsion

15. 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

16. Polony-FISSeq: up to 2 billion beads/slide Cy5 primer (570nm) ; Cy3 dNTP (666nm) Self Organizing Monolayer Jay Shendure

17. Polony FISSeq Stats • # of bases sequenced (total) 23,703,953 • # bases sequenced (unique) 73 • Avg fold coverage 324,711 X • Pixels used per bead (analysis) ~3.6 • Read Length per primer 14-15 bp • Insertions 0.5% • Deletions 0.7% • Substitutions (raw) 4e-5 • Throughput: 360,000 bp/min • Current capillary sequencing 1400 bp/min • (600X speed/cost ratio, ~$5K/1X) • (This may omit: PCR , homopolymer, context errors) Shendure

18. Anonymity, privacy, identity Required disclosure > optional > required privacy

19. Non-anonymous healthy genotype-phenotype studies • Are information-rich resources (e.g. facial imaging & • genome sequence) really anonymous? • What are the risks and benefits of "open-source"? • What level of training is needed to give informed consent on • open-ended studies? • Harvard Medical School IRB Human Subjects protocol • submitted 16-Sep-2004

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