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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
2 2
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
3
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
5
Genomes = Traits
TRAITS (Phenome)
PERSONAL GENOME 3M alleles
Standards and improved QC: Cohorts approved for global & commercial sharing
6
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
9
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
12
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
18
- - - - - - 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
19
- - - - - - 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: $2M
0.1 0.01
bp/$
20
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
21 21
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
22
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)
24 24
Why long haplotypes -- gaps in the reference genome
Reich et al. Nature Genetics 2005
Multiple Sclerosis
20Mb gap
Stretched DNA Fiber FISh/FISSEQ 3Mbp
26
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
29 29
Personalized organs-on-chip
Huh, Ingber et al. Science. 2010 Trends in Cell Biol 2011
+ neural, blood-brain-barrier, skin, testis
30
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
35 35
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
37 37
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
.
.
40 40
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
41 41
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