What’s Next in Next-Generation Sequencing?
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Dr. George Church Professor, Department of Genetics Harvard Medical School Dr. George Weinstock Professor of Genetics and Molecular Biology Washington University Dr. Joel Dudley Assistant Professor of Genetics and Genomic Sciences and Director of Biomedical Informatics Mount Sinai School of Medicine Webinar Sponsors What’s Next in Next-Generation Sequencing?
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What’s Next in Next-Generation Sequencing?. Webinar Sponsors. 1965 1D-RNA sequence Holley, Everett , Madison, Zamir. 2013? Complete 1D genome? 3D-genome? 3D-transcriptome?. 60 yr + 3D. 1953 3D-generic DNA Franklin, Gosling, Watson, Crick, Wilkins, Stokes , Wilson. - PowerPoint PPT Presentation
Transcript of What’s Next in Next-Generation Sequencing?
Dr. George Church Professor, Department of Genetics Harvard Medical School
Dr. George Weinstock Professor of Genetics and Molecular Biology
Washington University
Dr. Joel DudleyAssistant Professor of Genetics and Genomic Sciences and Director of Biomedical Informatics
Mount Sinai School of Medicine
Webinar Sponsors
What’s Next in Next-Generation Sequencing?
60 yr + 3D
19533D-generic DNAFranklin, Gosling, Watson,Crick,Wilkins, Stokes, Wilson
19651D-RNA sequenceHolley, Everett, Madison, Zamir
19773D-RNARich, et al.Klug, et al.Kim, et al.
2013?Complete 1D genome?3D-genome?3D-transcriptome?
1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 2015 2020 2025 2030 2035 20401E+3
1E+4
1E+5
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1E+8
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1E+13
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From $3 billion to an affordable genome
When? DNA sequencing& Moore's law
1.5x/yr for electronics
Optimistic’ exponential extrapolation
6 decades
1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 2015 2020 2025 2030 2035 20401E+3
1E+4
1E+5
1E+6
1E+7
1E+8
1E+9
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From $3 billion to an affordable genome
When?
6 years
DNA sequencing& Moore's law
1.5x/yr for electronics
Early arrival
Sequencing via Imaging. Clinical AccuracyHaplotype phase: 2 mutations in cis vs trans
1.43 Mbp LFR CGI/Harvard: Peters, et al. Nature July 2012
10 cells 384 aliquots, ~200 kb size 1 error per 10 million bp (Q70) ! Accurate genome = only 4 Mbytes.
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1988-1995: Church, Deamer, Branton, Baldarelli, Kasianowicz.
2009 Clarke, Bayley, et al 2010 Derrington, Gundlach, et al 2012 Cherf, Akeson, et al
Nanopore : Polymer vs Monomer vs NanoTag
GG AAA TTT CCC
2012: Oxford & Genia
5 GCAACAGAGCCAGC CCC ′ GCAACAGAGCCAGC AAA GCAACAGAGCCAGC CCC GCAACAGAGCCAGC TTT GCAACAGAGCCAGC GG A A15 3 .′
Nanopore: Polymer vs Monomer vs NanoTag
PEG-Labeled PP: Ju, Kasianowicz, et al. Scientific Reports 2012
“accuracy better than 1 in 5x108 events”A
C G T
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Fluorescent in situ Sequencing (FISSEQ)60 cycles x 4 colors (3D omes)
Lee, Yang, Terry, Nilsson, Church et al.
Single base differences
Danaher Polonator 2013: Automation & Image Processing
DNA ISH probe
USER cleavage
Probe extension with aminoallyl dUTP
Probe immobilization using BS(PEG)9
••••••••••••••••••••••••••aUaU
RNA
aU aU
RCA primer
aUaU
Phi29
RCA
ISH probe trimming to prevent self-
circularization
RCA with aminoallyl dUTP
Amplicon immobilization using
BS(PEG)9
Fluorescent probe hybridization or
sequencing
aU aU
ssDNA intramolecular circularization
RCA primer hybridization 0 10 20 30 40 50 60
02468
101214
Signal/BackgroundAbs Intensity
Re-probing cycles
RFU
0 1 2 30
100
200
300
400
500
Displacement (um)
Freq
uenc
y
AGCT
6 base sequencing(1 z-slice shown)
Sequencing-by-ligation(1 z-slice shown)
5’ phos
U Nu
CyNu
Cypmpm
pm
nm
Lee, Terry, Daugharty, Turczyk, Scheiman, Yang, Li, Nick Conway; Collaborators --- Kun Zhang, Angela de Pace lab, Feng Zhang, Nilsson
All RNAs GAPDH
Human fibroblast
How can we improve genome interpretation?
How we can make human omics data and cells shareable world-wide?
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Genomes + Environments = Traits
TRAITS(Phenome)
PERSONAL GENOME3M alleles
Nutrition
Chemicals
Immunome Stem-cellsEpigenome (RNA,mC)
Cancer
PersonalGenomes.org(US, Canada, UK, EU goals: 100,000 volunteers each)
Microbiome
Therapies
Immunome
evidence.personalgenomes.org
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GenomesEnvironmentsTraits (GET)• World’s only open access data sets• Consented for re-IDentification • 100% on Exam – Educate first• Stem Cell Biobank
0431
1070
1660
1677
1687
1833
1846
1731
1730
1781
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NIST + FDA + PGP Genome Standards
genomeinabottle.org
“enthusiastic about using samples from the Harvard University's Personal Genome Project, which are broadly consented”, Salit said.
Re-identification & commercial use
http://www.genomeweb.com/sequencing/nist-consortium-embarks-developing-meter-stick-genome-clinical-sequencing
N=1, Genome actionability
Volker: Intestinal surgery XIAPCord blood
Beery twins: Cerebral palsy SPRDiet 5HTP
Wartman: Leukemia FLT3Sunitinib
Gilbert: Healthy BRCA Mas/Ovarectomy
Snyder#82: Healthy GCKR, KCNJ11Diet, exercise
Smarr#74: Crohn’s IL23R Diet, probiotics
Bradfield: Healthy CDH1 GastrectomyPGEd.org 10/18
SupercentenarianStudy.comrare protective alleles
Mortensen 115.7
Breuning 114.6
Calment 122.4
Mirabella 110.6
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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 ~0, 1%•FUT2 -/- Stomach flu resistant 20%•APP -/+ Alzheimer’s 0.4%
blog.personalgenomes.org
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N=1, Cause Cure
"Long-Term Control of HIV by CCR5 Δ32/Δ32 Stem-Cell Transplantation" 2009 New England J Medicine
2012: Sangamo Phase 2 clinical trial
2007 Leukemia & AIDS:Timothy Ray Brown
Cas9 CRISPR
RNA-guided human genome engineering. Science in pressMali, Yang, Esvelt, Aach, Guell, DiCarlo, Norville, Church
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Brain Activity Map
(BAM+PGP)
GenesEnvironments
Traits
PersonalGenomes.org
Biswal etal PNAS 2010
Dr. George Church Professor, Department of Genetics Harvard Medical School
Dr. George Weinstock Professor of Genetics and Molecular Biology
Washington University
Dr. Joel DudleyAssistant Professor of Genetics and Genomic Sciences and Director of Biomedical Informatics
Mount Sinai School of Medicine
Webinar Sponsors
What’s Next in Next-Generation Sequencing?
