Fri 16-Nov-2007 DNA & Personalized Medicine:Being part of the big picture, Toronto

Thanks to:

Personal Genome Project

ABI, Illumina, Helicos, CGI, IBS, 454, Enzymatics, 23andme, Knome, DanaherMotion

PGP Volunteers ICPS

What if a government required testing of babies for intelligence genes?

PKU Phenylketonuria(Phenylalanine hydroxylase deficiency)Tested in nearly all 4M newborns per yearin the US (1 /15,000 births)

Close to 100% heritable (& 100% environmental)Nutritional preventative

Cancer predisposition

Risk reduction, after bilateral prophylactic mastectomy, of 90–100% -- Hartmann et al. J. National Cancer Institute, 2001

#13 #17Chromosome

BRCA-2

BRCA-1

Diagnosis : Sequence (not SNP chips)

Personal health citzenship

Huntington's ChoreaNancy Wexler’s family

AdrenoleukodystrophyAugusto Odone’s son

DougMelton’s son, Sam, has diabetes

Parkison’sDiseaseMichael J. Fox

Cancer, substance

abuseBetty Ford

Hugh Rienhoff

3D Facial Diagnostics

Control

22q11DS

Noonan

Smith-Magenis

William’s

Hammond et al, Am J Med Genet 2004, Am J Hum Genet 2005

Is anonymity in genomics realistic? http://arep.med.harvard.edu/PGP/Anon.htm(10) Re-identification after “de-identification” using other public data. Group Insurance Commission list of birth date, gender, and zip code was sufficient to re-identify medical records of Governor Weld & family via voter-registration records (1998) (9) Hacking. A hacker gained access to confidential medical info at the U. Washington Medical Center -- 4000 files (names, conditions, etc, 2000)(8) Combination of surnames from genotype with geographical infoAn anonymous sperm donor was traced on the internet 2005 by his 15 year old son who used his own Y chromosome genealogy to access surname relations.(7) Inferring phenotype from genotype Markers for eye, skin, and hair color, height, weight, racial features, dysmorphologies, etc. are known & the list is growing.(6) Self-identification. An example of this at Celera undermined confidence in the investigators. Kennedy D. Science. 2002 297:1237. Not wicked, perhaps, but tacky.(5) A tiny amount of DNA data in the public domain with a name leverages the rest. This would allow the vast amount of DNA data in the HapMap (or other study) to be identified. This can happen for example in court cases even if the suspect is acquitted.(4) Laptop theft. 26 million Veterans' medical records, SSN & disabilities stolen Jun 2006. (3) Unauthorized access to DNA bearing samples (e.g. hair, dandruff, hand-prints, etc.) (2) Identification by phenotype. If CT or MR imaging data is part of a study, one could reconstruct a person’s appearance . Even blood chemistry can be identifying in some cases. (1) Government subpoena. False positive IDs can be very disruptive.

PersonalGenomes.org Project Goals1) Active subject participation, informed redactions2) Entrance exam to ensure highly informed consent3) Subject & public access (not just research elite) 4) Eliminate over-promising de-identification5) Scaleable to millions of research subjects,

$1K DNA + trait data, donations6) Highly integrated, holistic, systems-biology

7) Subjects informed enough to “opt-out”8) Life-impacting info can reach subjects9) Cells available for personal functional genomics

0431

1070

1660

1677

1687

17311781

1833

1846

Reducing costs via open-sourcehardware, software, wetware

Factor • 30 Equipment speed: from 1 up to 30 Mpixels/sec camera• 4 Equipment cost: from $500K down to $124K (Danaher Inc)• 36 Parallelism: 36 flow-cells per camera, 60M beads each

------------------• 75 Flow cell volume: 1.5 mm down to 0.02 mm• 40 Kit costs: $2000 down to $50 at standard enzyme costs• 10 Enzymes: $4000/mg down to <$400 (Enzymatics Inc)• 50 Genomic subset enrichment (Exome – 1% genome)

PGP-HardwareNikon, Hamamatsu, Alcott, Prior, Sutter, Bioptechs, PI, Uniblitz, Superlogics

Shendure, Porreca et al. 2005

Danaher, Hamamatsu, Nikon$124K with computer & 1 yr serviceRich Terry et al. 2007kevin.mccarthy@danahermotion.com

DIY

36 Flowcells

• Ultra-clean conditions for reduction of background amplification + Real-Time monitoring

• Post-amplification chip hybridization distinguishes alleles

• Amplification variation random & easily filled by PCR

• error rate <1.7 10–5

Single-cell or Single DNA-fragment (haplotype) sequencing: 5 Mbp

Zhang et al. Nature Biotec 2006

Rearrangements detected using polony paired end reads Shendure et al Science Sep 2005

Deletion Insertion Inversion(rare in this clonal population)

Circle-capture 1% genome

3,5: duplicate controls

(no genome)

Porreca et al. Oct 2007 Nature

Methods

Oct 2007 R=.96

Jin Li et al. unpublished

Gapfill

Genomic DNA

Lymphocyte

cDNA

Fibroblast

cDNA

Keratinocyte

The other 99%? Allele-specific expression

T/C = 0.51 T/C = 3.47

Zhang et al. unpublished

PGP Cell lines from Coriell.org

ATP synthase

Writing to genomes: Personal Genome Expression multiple cell-types from accessible adult cells

Viral delivery: β-catenin, Dnmt3l, Dppa2,3,4,5, Ecat1, Eras, Fthl17, Grb21, Ecat8, Fbx15, Gdf3, hTERT, Klf4, Myc, Nanog, Oct4, Rex1, Sall4, Sox2, Sox15, Stat3, Tcl1, Utf1Signals: Activin, BMP4, Estrogen, FGF, HGF, PDGF, Retinoate, Shh, TGF, VEGF.

Human fibroblast derivatives - J.Lee(ecto-, endo-, meso- dermal markers)

Open-source hardware, software, wetware, ELSware

Reading genomes• Haplotyping & single-cell microbiome• Paired-end-tags (PET) for rearrangements • Selected genome subsets• Allele-specific expression• Bio-weather-map: Viral, allergen, VDJ-ome• ELS: PGP low cost, education for genome/trait sharing

Writing genomes• Re-programming adult human cell types• Combinatorial, automated allele replacements

.