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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

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personal genomes medicine
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

why sequence
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.

the idea of common snps for common diseases has been hugely oversold
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?

rare alleles common diseases
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.

personal genomics cancer therapy
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.

why single molecule sequencing
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)

cd44 exon combinatorics zhu shendure
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.

cd44 rna isoforms
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.

slide9

Multi-locus haplotyping on

pooled samples

Throughput = (# loci × # samples) / time

Kun Zhang

multi locus haplotyping
Multi-locus haplotyping

NOS3

Chr 7

C/T

G/A

G/T

G/A

T/A

C/T

C/T

~24-Kb

chromosome wide haplotyping

IL6-3572 : A/C

~60-Mb

CD36-4366 : T/A

Chromosome-wide haplotyping

Human Chr. 7

A..A

A..T

convergence on non electrophorectic tag sequencing methods
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

slide13

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

polony f luorescent i n s itu seq uencing libraries

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

cleavable dntp fluorophore terminators
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

slide16

Polony-FISSeq: up to 2 billion beads/slide

Cy5 primer (570nm) ; Cy3 dNTP (666nm)

Self Organizing Monolayer

Jay Shendure

slide17

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

anonymity privacy identity
Anonymity, privacy, identity

Required disclosure > optional > required privacy

non anonymous healthy genotype phenotype studies

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