Thanks to: .gov || || .edu || || .org || || .com || || Read = = = = = = = = I/O = = = = = = = Write 1

Standards & Open-Access Genome-Environment-Trait Data NIST 10:15-10:45 AM 16-Aug-2012

Azco

ArmRev.org Oppenheimer Foundation

Gen9

LSRF

NHGRI NIGMS

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Technology & Genome Standards

(1) Reference Material : cell lines, primary cells, synthetic DNA spiking

(2) Sequencing: Haplotype, nanopore, in situ other methods: Forensic, immune DNA (3) Bioinformatics, Data Integration, & Data

Representation: methods to analyze & integrate the data

(4) Performance Metrics & Figures of Merit

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Individually Rare -- Collectively Common (10%)

2443 diseases (~6000 genes) are highly predictive & medically actionable

1963 PKU 1991 BRCA 2010 HCM

Genetests.org PGEd.org

Case studies

Nic Volker: not intestinal surgery à cord blood Beery twins: not cerebral palsy à Diet 5HTP Dr. Lukas Wartman: Leukemia à Sunitinib Ivacaftor: treat CFTR G551D à 3 months in FDA

PGEd.org

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Genomes = Traits

TRAITS (Phenome)

PERSONAL GENOME 3M alleles

Standards and improved QC: Cohorts approved for global & commercial sharing

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Genomes + Environments = Traits

TRAITS (Phenome)

PERSONAL GENOME 3M alleles

Food Metabolome/Tox

Immunome Epigenome RNA,mC Proteome

PersonalGenomes.org

Microbiome

Therapies

Immunome

4D-Imaging Stem-cells

Cancer

Standards and improved QC : Cohorts approved for global & commercial sharing

Genomes + Environments = Traits

PersonalGenomes.org US, Korea, Israel, Germany, Canada

Individuals willing to have their genomes, cells (saliva, blood, skin, iPS), extensive trait data Open-access : CC0 16K volunteers registered in 74 countries Harvard IRB approval for 100K 2,418 achieved 100% on entrance exam

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Wireless environment, drug & physiology monitors

Kim et al Science 2011, GE Vscan ultrasound, Piix cardiac monitor

PGP#1

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Rare Protective alleles e.g. Myostatin

Enhanced muscle growth, decreased body fat & decreased atherosclerosis (2009 Endocrine Society, Bhasin, et al BU)

Flex Wheeler

MSTN -/-

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Correlation → Cause → Cure/Prevention Rare Protective alleles

• MSTN -/- Lean muscles <0.001% • LRP5 -/+ Extra-strong bones 0.001-8% • PCSK9 -/+ Lower coronary disease 3, 0.06% • CCR5 -/- HIV-resistant (Pox/Plague) ~0, 1% • FUT2 -/- Stomach flu resistant 20% Embrace the extremes: informative, easy, powerful

blog.personalgenomes.org

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Precise Genome Therapy: prevent/cure HIV

"Long-Term Control of HIV by CCR5 Δ32/Δ32 Stem-Cell Transplantation" 2009 New England J Medicine

Sangamo Phase 2 clinical trial

U.S. District Court rules that stem cells are drugs

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Microbiome & Immunome Impact Metabolome

Microbe tests: Detect Drug resistance spectrum Earlier warning (e.g. meningitis) Immune tests: Focus on response to exposure Longer times to detect exposure (e.g. HIV, TB)

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PGP Immunome time series

Harvard/MIT: Vigneault, Laserson, Lieberman-Aiden, Church Roche: Egholm, Simen

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Rare (therapeutic) antibodies

Broadly reactive antibody … potent neutralization of HIV-1 … unusually long, 28-amino acids (84 bp), CDR3… towers above the antibody surface. Pejchala et al. PNAS 2010

Antibody-based protection against HIV infection by vectored immunoprophylaxis Balazs, Baltimore et al. Nature 2012

RNAi & Drug alternative #2

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PGP CDR3 size distribution

Laserson, Vigneault

84 bp

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PGP Vaccination 3 year time series.

-8 -2 -0.04 1 3 7 14 21 28 days

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Proteome Antigen Libraries

Larman et al Nature Biotech 2011

Human, bacterial, viral, food, allergens

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- - - - - - Moore’s law 1.5x/yr for electronics

$1000 Genome

When?

2040

2004-6: $400M

2000-4: $3 billion

0.1 0.01

bp/$

2015 2020… 2025 2030 2035 2040

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- - - - - - Moore’s law 1.5x/yr for electronics

Factors of

10/yr

$1000 When?

2012

2011 $4K

2004-6: $400M

2000-4: $3 billion

2012 $0.8K

2007: $2M

0.1 0.01

bp/$

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How? Next-generation technologies 1. Polonator MA 2. Roche-454 CT 3. AB-SOLiD MA 4. Illumina UK,CA 5. CGI CA 6. Helicos MA 7. Pacific Bio CA 8. IntelligentBioSys MA 9. Ion Torrent CT 17. LightSpeed CA 10. Genapsys CA 11. Electronic Biosci CA 12. Nabsys RI 13. OxfordNanopore UK 14. IBM-Roche NY 15. NobleGen MA 16. Genia CA

18. GnuBio MA 19. Bionanomatrix PA 20. Halcyon CA 21. ZS Genetics NH 22. Electron Optica CA 23. Genizon BioSci QC 24. LaserGen TX 25. GE Global NY 26. Stratos Genomics WA 27. Reveo NY 28. Firebird FL 29. Zeiss MA 30. Lucigen WI 31. Adv. Liquid Logic NC 32. Caerus Molec Diag CA

http://arep.med.harvard.edu/gmc/nexgen.html

33. Nanophotonics Biosci CA 34. Network Biosystems MA 35. SeiraD NM 36. Affymetrix CA 37. Population Gen Tech UK 38. AQI Sciences AZ 39. Base4innovation UK 40. Li-Cor NE 41. U.S. Genomics MA 42. Mobious Genomics UK 43. Visigen TX 44. Starlight CA

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Nanopore : Polymer vs Monomer

ONT & Genia 1995: “use a polymerase … while recording conductance changes” Church, Deamer, Branton, Baldarelli, Kasianowicz.

2009 Clarke, Bayley, et al 2010 Derrington, Gundlach, et al 2012 Cherf, Akeson, et al

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2012 Sequencing ONT/Genia Danaher/IBS $/device 0-30K 150K $/PG 100k-2K 1K Read length 100K 70 Speed (days) 0.1 30 Size (kg) 0.2 50 Sorting No Yes In situ No Yes

http://arep.med.harvard.edu/gmc/nexgen.html

June 2012

Clinical Importance of Haplotype vs WGS/Exome 2 mutations in cis vs trans!

386 kb fosmid Kitzman, et al Nat Biotech 2011 1429 kb LFR CGI Peters, et al. Nature July 2012 65pg =10 cells à 60-300 kb in 384 aliquots. 1 false positive SNV per 10 Mb. (Q70)

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Why long haplotypes -- gaps in the reference genome

Reich et al. Nature Genetics 2005

Multiple Sclerosis

20Mb gap

Stretched DNA Fiber FISh/FISSEQ 3Mbp

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Zhang et al Nature Gen 2006 Mitra & Church NAR 1999 (FISSEQ)

In Situ Sequencing: metaphase haplotypes

Rare cells: Resistance in Leukemia ABL Tyr-Kinase Nardi, Raz, Chao, Wu, Stone, Cortes, Deininger, Church, Zhu, Daley. Oncogene

E255K

T315I

M244V

Personal Genome Project & Biobanks: iPS (with Coriell)

iPS-derived teratoma Endoderm

Ectoderm Mesoderm

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Personalized organs-on-chip

Huh, Ingber et al. Science. 2010 Trends in Cell Biol 2011

+ neural, blood-brain-barrier, skin, testis

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Read: Fluorescent in situ Sequencing (FISSEQ) 60 cycles x 4 colors

Lee, Yang, Terry, Nilsson, Church et al.

Single base differences

Epigenom, Transcriptome in situ

1. Fix cells 2. Reverse transcribe 3. Cross-linking cDNA 4. RNA digestion 5. Enzymatic circularization

Fluorescent In situ Sequencing (FISSEQ)

In situ sequence bar-coded FISH probe sequencing using confocal microscopy in human fibroblasts

3-D reconstruction of in situ RNA-seq in human iPS cells showing DMNT3b, GAPDH, EEF1alpha and GAL

Signal to noise ratio remains stable over 60 cycles

Reference Cycle 1

(Richard Terry and Chao Li)

Cycle 25 Cycle 50

iPS FISSEQ (Manual cycling)

Probe hyb

Probe strip

(average of 10 spots; 3 pixel x 3 pixel area per spot from 20x epifluorescence imaging)

Overcoming the imaging resolution barrier #1

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Super-resolution #2: Polonies beads & Rolony grid

Synthetic Aperture Optics

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Challenge of QC of (epi) genetic programming

72/101 Non-silent changes in 20 hiPS cell lines Not random mutations during reprogramming (p < 8E-50) Gore, Zhang, et al. Nature. ABCA3, AKR1C4, ANKRD12, ANKRD12, ARHGEF5, ASB3, ATM, C14orf174, C1orf100, CABC1, CACNG3, CALN1, CARM1, CCKBR, CELSR1, DLG3, DNAH3, DSC3, DYNC1H1, FAT2, GDF3, GOLGA4, GSG1, GTF3C1, HK1, HK1, IFNGR1, IFT122, INTS4, IQGAP3, ITCH, KLRG2, LINGO2, LRP4, MARCKSL1, MMP26, MYRIP, MYRIP, NEK11, NEK5, NTRK3, NTRK3, OR6Q1, OSBPL3, PBLD, POLE, POLR1C, PPP1R2, PRICKLE1, PTPRM, RANBP3L, RASEF, RFX6, RGS8, RP4, SAL1, SCN1A, SCN1A, SDR16C5, SEMA6C, SH3PX3, SLC1A3, SLC1A3, SORCS3, SPATA21, SPEN, TM9SF4, TMEM40, TNR, UBA2, VAC14, VMO1, ZER1, ZNF16, ZNF471, ZZZ3

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Technology & Genome Standards

(1) Reference Material : cell lines, primary cells, synthetic DNA spiking

(2) Sequencing: Haplotype, nanopore, in situ other methods & validation: (3) Bioinformatics, Data Integration, & Data

Representation: methods to analyze and integrate the data

(4) Performance Metrics & Figures of Merit

.

.

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1977 cm Q20 2012 600 nm Q70 55 bp 1000 genomes /month 50X 6Gbp CGI

Drmanac et al. Science 2009

nobelprize.org/nobel_prizes/chemistry/ laureates/1980/gilbert-lecture.pdf

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Sequencing Technology: Next In Situ - Clinical - Portable

Hairy =red, Kruppel=green, Giant=blue. Kozlov et al In Silico Bio 2002

Oxford Nanopore Genia

Lauerman, Bloomberg Intelligent BioSystems

Lauerman, Bloomberg In situ Sequencing

John Lauerman (PGP#16) JAK2 polycythemia vera, essential thrombocythemia, myelofibrosis

Steve Pinker (PGP#6) HCM

DNA Explorer (Ages 10 and up)

DIY Bio

OCTOBER 1, 2010 Obsessed With Genes (Not Jeans), This Teen Analyzes Family DNA

PGP#14 John West Factor V Leiden