Thanks to:

.gov||||.edu||||.org||||.com|| ||

Read = = = = = = = = I/O = = = = = = = Write 1

Personal Genome Project 9:35-10:20 Hyatt 4-Oct-2010 Future of Healthcare Using Genomics as a Key Tool

AzcoRBH

ArmRev.orgOppenheimer Foundation

Gen9

LSRF

22

Will we be able to handle & interpret the data?

Christley et al Bioinformatics

(2009) apply a series of techniques to James Watson's genome that in combination reduce it to a mere 4MBytes, small enough to be sent as an email attachment. algorithms implemented in C++ and are freely available http://www.ics.uci.edu/~xhx/project/DNAzip

(Google processes 24E9 Mbytes/day)

3

Medical Genomics: Individually rare collectively common (10%)

1935 genes are highly predictive

& medically actionableat ~$1000 per gene test.

Why not on DTC SNP chips? Cost? Patents?PKU, Tay Sachs, Cystic Fibrosis, BRCA1/2, etc.rarediseases.info.nih.gov genetests.org

1963 PKU

44

US clinics quietly embrace whole-genome sequencing

Nature 14-Sep-2010

"If one hospital is doing it, you can be sure others will start, because patients will vote with their feet," --

Elizabeth Worthey HMGC & Children's Hospital of Wisconsin

“At the age of 3, he had more than 100 separate surgeries …On the basis of [sequencing], the physician recommended a bone-marrow transplant in June 2010. By mid-July, the child was eating his first meal.”

5

Reading & Writing Genomes: First semi-synthetic plasmid 1978: $10/b

CGI Human diploid genome 2009: $1500 / 6Gb

pBR3225 genes

(=7 logs/30y mostly since 2005)

NAR 1978Sutcliffe & Church(BR:Bolivar & Rodriguez)

•Human insulin•Human growth hormone•Alpha-interferon•G-CSF•TPA•GM-CSF•Gamma-interferon•IL-2•Erythropoietin•Hepatitis B vaccine

(Amgen, Biogen, Genentech, etc)

6

0.000001

0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

10000

100000

1000000

1980 1990 2000 2010

dsDNAOligosSeq bp/$

NIBR SynBio 15-Oct-2010

George Church

1Mb/$ 0.1Mb/$ chip

Moore’s law =1.5x/yr vs 10x/yr

1st-generation Gene synthesis vs

2nd-generation Sequencing (21 tech)

& DNA synthesis

5-log GAP

1b/$in cells

7

2nd-generation sequencing technologies1.

Illumina-GA SbP Fluorescent read-length (2*110 bp)2.

AB-SOLiD SbL Longest ligation reads (2*70 bp)3.

CGI

SbL $2000 genome, rolony grid, (7*10bp)4.

Polonator

SbL/P

Open-source,

$170K device, (2*30bp)5.

Roche-454 SbP Long reads (900 bp))6.

Helicos SbP-sm

High parallelism & quantitation (2*30 bp)7.

Ion Torrent SbP

$50K, small device (100 bp)8.

Pacific Bio SbP-sm

Long reads (>2.0 kb)9.

Intelligent Bio

SbP hexagonal grid (2*30 bp)10. GnuBio

SbP-picoliter droplets (ND)11.

Halcyon EM-sm

Long reads (ND >Mb)12.

Visigen/StarLite SbP-sm

Qdot-Pol-dNTP FRET (ND 500Kb)13.

Bionanomatrix SbP-sm

Fluorescent mapping (ND)14.

OxfordNanopore

Pore-protein-sm

small device (ND)15.

Nabsys

Pore-SbH-sm

small device (ND)16. IBM

Pore Si-sm

small device (ND) 17.

Genizon BioSci

SbH in situ sequencing (ND)18. LightSpeed

SbL 16X density, >10X speed (ND)19.

ZS Genetics

EM-sm

Iodine labels (ND)20. GE Global

SbP-sm

(ND)21. Electronic Biosci

Pore-protein-sm (ND)

Polonator Polonator

Beadless Isothermally-amplified Rolling Circle Molecules on a grid

8•Billions of Library Molecules can be amplified in 1 ml of solution in 30 minutes•Unamplified molecules do not take up flow-cell space

Polony Exclusion Principle

9

Human Genome Sequencing Using Unchained Base Reads on Self-assembling DNA Nanoarrays.

Drmanac et al Science Jan 2010

X,Y density: 5-fold due to grid * 25 vs 1 pixel/template

Nutrition

Chemicals

10

Genomes Environments

Traits

Immunome TRAITS(Phenome)

Personal stem-cells

Epigenome (RNA,mC)

PERSONAL GENOME

3M alleles

One in a life-time genome + yearly ( to daily) testsBio-weather map : Allergens, Microbes, Viruses

PersonalGenomes.org

Microbiome

Therapies

Immunome

Value of extreme & distinctive traits

Hyperthymesia, savant, synaesthesiaSolomon Shereshevskii 1886-1958 Kim Peek 1951-2009Brad Williams 1956-present

PhysicsIsaac Newton 1643-1727Albert Einstein 1879-1955

SpiritualSiddhārtha Gautama 563-483 BCETenzin Gyatso 1935-present

1212

2 Alleles from 4 Genome Sequences

MYH3 R672H R672C Ng et al Nature 20009Freeman–Sheldon syndrome (FSS, dominant)

1313

3 Alleles from 2 Genome Sequences

Whole-Genome Sequencing in a Patientwith Charcot–Marie–Tooth NeuropathyLupski et al. NEJM 362;13 April 1, 2010SH3TC2 R954X, Y169H

Genetic diagnosis by whole exome captureand massively parallel DNA sequencing.Choi, Lifton, et al. PNAS 2009“Homozygous missense D652N mutation at a position in SLC26A3 (congenital chloride diarrhea locus)”

1414

4+10 Alleles from 4 Genome Sequences

Analysis of Genetic Inheritance in a Family Quartet by Whole- Genome Sequencing. Roach, et al. Science 2010

Exome Sequencing Identifies the Cause of a MendelianDisorder. Ng et al. Nature Gen. 2010

• Primary ciliary dyskinesia (lungs)DNAH5• Pyrimidine synthesis (face & limbs) DHODH

Logan & Heather Madsen

sltrib.com/health/ci_14648608

15

Genes Environments

Traits, cells1) First/only open access data 2) Avoid over-promising on de-identification 3) 100% on

Exam

to assure informed consent

(*Educate pre-consent rather than post-discovery*)4) Genome sequence

+ epigenome

5) Multi-traits: images, iPS-etc.RNA, microbe/VDJ 6) Cells available

for personal functional genomics

7) IRB approval for 100,000 diverse volunteers501(c)(3)

16,000 so far

20431

21070

2166

0

21677

21833

21687

21846

21731

21730

21781

Genome+Environment = Traits, [diseases], (treatments)

Hair: Baldness [alopecia](minoxidil) Eyes:

[Near/Far-sightedness](glasses) Iris color [ARMD] (sunglasses)Face:

[Developmental syndromes, Wrinkles] (Botox)Brain:

ADHD(Ritalin); Depression(Prozac); Headache(analgesics)Sleep & Circadian (caffeine, amphetamine, modafinil)Motion sickness (Dramamine, and Scopolamine)Ears:

Sensitivity (hearing aids)Nose:

Shape [breathing disorders] (CPAP)Lip:

[Cleft palate] (surgery); [Hirsutism] (calcium thioglycolate)Mouth: Halitosis, throat exams; aerosols [airborne pathogens]Digestion [reflux, gas,ulcer] (antibiotics, antacids, PPIs) Back: Strain sensitivity [IDD] (analgesics)Skin:

Perspiration, Body odor, Pheromones (deodorants)Surface texture [psoriasis] (topicals, photo-treatments)Immune components [acne] (topical antibiotics) Skin color [vitamin D & sunburn] (supplements, SPF cream)Hands: Dermatoglyphics [syndromes], [Arthritis](corticosteroids) Internal sensors:

Proprioceptor, Repetitive stress (NSAIAs)Body: Height [Marfan] [short stature] (hGH)Weight [anorexia] [obesity] (Orlistat, Phentermine, Sibutramine)Allergies (antihistamines, cortisone, epinephrine, theophylline)Metabolic polymorphisms (nutriceuticals, insulin, statins)Feet:

Plantar fasciitis (orthotic shoes)Athlete’s foot (miconazole, itraconazole, terbinafine, salicylate)1933

PGP

1717

PGP#1 fMRI

Randy

Bruckner

Behavioral & cognitive

tests

Ken Nakayama

Prosopagnosia

1818

Individual report:

97 year old

PGP11

evidence.personalgenomes.org

1919

How do we improve interpretation? e.g. PGP 6 hypertrophic cardiomyopathy allele

evidence.personalgenomes.org

20

Microbiome vs

Immunome

Microbe tests: Detect Drug resistance spectrumEarlier warning (e.g. meningitis)

Immune tests: Focus on response to exposureLonger times to detect exposure (e.g. HIV, TB)

21

Microbiomes: What limits diagnostics?

-Standard practice: skip diagnostics; guess at pathogen & antibiotics-Biomarkers vs causative sequences.-Ideally target pathogenicity & resistance- Assay 6 nanoliters or 6 liters?

(if <1 cell / ml)

22Yung, Ingber et al. Lab Chip, 2009

Circulating tumor, pathogen, fetal, immune cells

23

Microbiome vs

Immunome

Microbe tests: Detect Drug resistance spectrumEarlier warning (e.g. meningitis)

Immune tests: Focus on response to exposureLonger times to detect exposure (e.g. HIV, TB)

24

Vaccination Immunome

Harvard/MIT: Vigneault, Laserson, Lieberman-Aiden, Church

Roche: Egholm, Simen

25

PGP Time Series Vaccine ExperimentTracking human dynamic response to vaccination to 11 strains:Hepatitis A+B, Flu A/Brisbane/59/2007 (H1N1)-like, 10/2007

(H3N2)-like, B/Florida/4/2006-like virus Polio, Yellow feverMeningococcusTyphoid, Tetanus Diptheria, PertussisCollect samples at -14d, 0d, +1d, +3d, +7d, +14d, +21d, +28d

26

SR1+SR2+TR1

IMGT/LIGM

V D J usage –

CDR3 size distribution

PGP Vaccination Immunome Self Organizing Map (SOM) clustering

27Synthesize and test for antigen binding.

Chemicals28

Genomes Environments

Traits

Immunome TRAITS(Phenome)

Personal stem-cells

Epigenome (RNA,mC)

PERSONAL GENOME

3M alleles

One in a life-time genome + yearly ( to daily) testsBio-weather map : Allergens, Microbes, Viruses

PersonalGenomes.org

Microbiome

Therapies

Immunome

29

PGP#1 & #9 skin to stem cells to ...

Lee J, Park IH,

Gao Y, Li JB, Li Z, Daley G,

Zhang K, Church GM

(2009)

A Robust Approach to Identifying Tissue-specific

Gene Expression Regulatory Variants Using Personalized Human Induced Pluripotent Stem Cells. PLoS Genetics Nov 2009

30

PGP iPSC-derived hepatic proteins & activity

Generation of Functional Human Hepatic Endodermfrom Human Induced Pluripotent Stem CellsGareth et al (Daley, Church, Ian Wilmut labs)

31

G

A

TC

Allele‐specific expression (ASE)

Test cis element

variants in heterozygotes

GA

AAAAAAAAAAAAAAAAAAAA

TC

TT

eliminate environmental & trans-acting variation among individuals.Cis:

Copy number, enhancer, promoter, splicing, polyA, termination,

transport, decay.

G

A

GG

Allele‐specific 

transcription factor 

binding

TF

Causality: 

Synthetic 

homologous 

allele‐

replacement

Digital RNA allelotyping

Zhang, Li, Church unpublishedForton et al. Genome Res. 2007

32

Clustering stat- significant

allele-specific expression in

reprogrammed cells, ~50% of ASE invariant

among cell types

LeeZhangParkDaleyChurch

33

In situ sequencing for metaphase chromsomes

Zhang et al 2006

In situ sequencing: Resistance to BCR-ABL kinase inhibitors in CML therapy Nardi, Raz, Chao, Wu, Stone, Cortes, Deininger, Church, Zhu, Daley. Oncogene 27:775-82

E255K

T315I

M244V

Bakal, et al. Science 316, 1753 35

145 morphological measures → in situ sequencing

3636

The Future of Reading & Writing Genomes

• Next Generation BI/O: Reading & WritingSafety: multivirus-resistance, Genomic isolation

•Personal Genomes –

Integration tasks- Personal Genomes, Environments, Traits, -

Stem cells, Microbiome/Immunome

•Causality: Multiplex changes innetwork causes & monitor effects.

•In situ multiplexing: subcellular, paralogs, alleles•Proteome: antibody multiplexing•Human diversity:

multiplexing

3737

.

38

Promising (implying) anonymity?

Research identification trend (12) Identify individual case/control status from pooled SNP data Homer et al

PLoS Genetics 2008

as this became known, NCBI pulled dbGAP data (11) Re-identification after “de-identification”

using public data. Group Insurance list of birth date, gender, zip code sufficient to re-identify medical records of Governor Weld & family via voter-registration records (1998)

Self identification trend (10) Unapproved self-identification. e.g. Celera IRB. (Kennedy Science. 2002)(9) Obtaining data about oneself

via FOIA or sympathetic researchers. (8) DNA data CODIS data in the public domain. even if acquitted

39

Anonymity vs Open Access? Are we in denial?Accessing “Secure data”(7) Laptop loss. 26 million Veterans' medical records,

SSN & disabilities stolen Jun 2006. (6) Hacking. A hacker gained access to confidential medical info at the U.

Washington Medical Center --

4000 files (names, conditions, etc, 2000)(5) Combination of surnames from genotype with geographical info

An anonymous sperm donor traced on the internet 2005 by his 15 year

old son who used his own Y chromosome data.

(4) 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.

(3) Inferring phenotype from genotype

Markers for eye, skin, and hair color, height, weight, geographical features, dysmorphologies, etc. are

known & the list is growing.

(2) “Abandoned DNA”

bearing samples (e.g. hair, dandruff, hand-prints, etc.) (1) Government subpoena. False positive IDs and/or family coercion

index

Sharing genetic information on the Web-- (Forbes 05.07.07 by Peter Huber

Manhattan Institute's Center for Legal Policy)

--will lead to cures. .. Routine lab tests .. already expose genetic variations associated with cholesterol, cancer and hundreds of other traits and diseases, both common and rare. Coming soon: .. dipsticks for the genes that control .. stress, pleasure, irritability, aggression, impulsive behavior, suicidal tendencies, alcoholism and sexual proclivities.. Discovering what ails us and how to beat it is a statistical game. .. traditionally .. left to .. Washington, because they had the best access to the most data. The networked masses, first to know what hanky-panky they've been up to, what pills they've been popping, and whether they feel better or worse. What Google medicine will lack in discipline and traditional rigor, it will more than make

up for in speed and scope.

Heritability & multigenics

Existing large studies Study Start Cohort size SamplesFramingham Heart Study '48 13,833 1,500Health Professional's study '86 52,000 30,000Nurses Health Study I '89 122,000 63,000Washington County study '89 33,000 33,000Women's Health study '92 40,000 28,000Women's Health Initiative '93 162,000 162,000NCI PLCO study '94 155,000 70,000Nurses Health Study II '96 116,000 60,000Am Cancer Soc CPS II-Lifelink study '98 184,000 109,000Multiethnic Cohort study '96 215,000 80,000VITAL cohort '99 78,000 54,000Agricultural Health study '99 90,000 35,000Southern Community Cohort study '02 90,000 80,000Black Women's cohort study '05 59,000 41,000

1,409,833 846,500

43

Trends toward opennessHR 2764

“SEC. 218. all investigators funded by the NIH submit ..

an electronic version of their final, peer-reviewed manuscripts .., to be made publicly available”

(make science paid for by tax-payers

accessible to the tax-payers, not just the experts) 26-Dec-07

PatientsLikeMe.com: MS, Parkinson’s, ALS, Depression, Anxiety, Bipolar, OCD, HIV/AIDS

“sharing your healthcare experiences and

outcomes is good.”

(Full names & photos)

44

Traits

Down Syndrome

Waardenburg

Eyes

Normal Dermatoglyph

45

Imaging Diagnostics

Control

22q11DS

Noonan

Smith-Magenis

William’s

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

46

How many ‘complete’

public genomes now?

Name >90% publicFold

coverage Inst TechVenter Sep-07 7.5 JCVI ABIWatson Apr-08 7.4 BCM 454Yang Nov-08 36 BGI ILMN

Kim 1(4) May-09 29 Incheon ILMNQuake Aug-09 28 Stanford HLCSChurch Nov-09 45-90 PGP CGIGates 2 Mar-10 20 PGP ILMN

Tutu Mar-10 30 PSU SOLiDAngrist Mar-10 30 PGP ILMN

Madsen 0(4) Mar-10 51-88 ISB CGILupski Mar-10 30 BCM SOLiDLucier Apr-10 ? ABI SOLiDFlatley Apr-10 ? ILMN ILMN

West 1(4) Apr-10 ? ILMN ILMNGill Apr-10 15 TBI/PGI ILMN

13