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 Dudley
Assistant Professor of Genetics and
Genomic Sciences and Director of
Biomedical Informatics
Mount Sinai
School of
Medicine
Webinar Sponsors
60 yr + 3D
1953
3D-generic DNA
Franklin,
Gosling,
Watson,
Crick,
Wilkins,
Stokes,
Wilson
1965
1D-RNA sequence
Holley,
Everett,
Madison,
Zamir
1977
3D-RNA
Rich, et al.
Klug, et al.
Kim, et al.
2013?
Complete 1D genome?
3D-genome?
3D-transcriptome?
Optimistic’ exponential extrapolation
From $3
billion to an
affordable
genome
DNA sequencing
1E+13
1E+12
1E+11
When?
1E+10
1E+9
1E+8
6 decades
& Moore's law
1.5x/yr for
electronics
1E+7
1E+6
1E+5
1E+4
1E+3
1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 2015 2020 2025 2030 2035 2040
3
From $3
billion to an
affordable
genome
Early arrival
1E+13
1E+12
1E+11
When?
1E+10
1E+9
6 years
1E+8
DNA sequencing
& Moore's law
1.5x/yr for
electronics
1E+7
1E+6
1E+5
1E+4
1E+3
1965 1970 1975 1980 1985 1990 1995 2000 2005 2010 2015 2020 2025 2030 2035 2040
4
Sequencing via Imaging. Clinical Accuracy
Haplotype 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.
Nanopore : Polymer vs Monomer vs NanoTag
1988-1995:
Church,
Deamer,
Branton,
Baldarelli,
Kasianowicz.
GG
TTT CCC
2012: Oxford & Genia
AAA
2009 Clarke, Bayley, et al
2010 Derrington, Gundlach, et al
2012 Cherf, Akeson, et al
5′ GCAACAGAGCCAGC CCC
6
GCAACAGAGCCAGC AAA GCAACAGAGCCAGC CCC
6
GCAACAGAGCCAGC TTT GCAACAGAGCCAGC GG A A15 3′.
Nanopore: Polymer vs Monomer vs NanoTag
A
C
“accuracy better than
1 in 5x108 events”
G T
PEG-Labeled PP: Ju, Kasianowicz, et al.
Scientific Reports 2012
Fluorescent in situ Sequencing (FISSEQ)
60 cycles x 4 colors
(3D omes)
Single base
differences
Lee, Yang, Terry, Nilsson, Church et al.
8
Danaher Polonator 2013:
Automation & Image Processing
Lee, Terry, Daugharty, Turczyk, Scheiman, Yang, Li, Nick Conway; Collaborators --- Kun
Zhang, Angela de Pace lab, Feng Zhang, Nilsson
ISH probe trimming to
prevent selfcircularization
ssDNA intramolecular
circularization
RCA primer
hybridization
RCA with aminoallyl
dUTP
U
DNA ISH probe
aUaU
5’
phos
Fluorescent probe
hybridization or
sequencing
pm
Cy
Nu
Cy
pm
Nu
nm
USER cleavage aU aU
RCA primer
aU
aU
14
12
10
8
6
4
2
0
500
Signal/Backgro
und
Abs Intensity
400
300
200
100
0
0
10 20 30 40 50 60
Re-probing cycles
0
1
2
aU
aU
Sequencing-byligation
(1 z-slice shown)
A
G
C
T
3
Displacement (um)
Phi29
RCA
Amplicon
immobilization using
BS(PEG)9
pm
Frequency
Probe immobilization
using BS(PEG)9
RNA
••••••••••••••••••••••••••
RFU
Probe extension with
aminoallyl dUTP
6 base sequencing
(1 z-slice shown)
Human fibroblast
All RNAs
GAPDH
How can we improve genome
interpretation?
How we can make human omics data and
cells shareable world-wide?
Genomes + Environments = Traits
PersonalGenomes.org
(US, Canada, UK, EU goals: 100,000 volunteers each)
evidence.personalgenomes.org
Therapies
PERSONAL
GENOME
3M alleles
Immunome
Immunome
Stem-cells
Epigenome
(RNA,mC)
Cancer
TRAITS
(Phenome)
Microbiome
Chemicals
Nutrition
13
Genomes
Environments
Traits (GET)
•
•
•
•
World’s only open access data sets
Consented for re-IDentification
100% on Exam – Educate first
Stem Cell Biobank
0431
1660
1070
1846
1677
1730
1731
1687
1833 1781
14
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-developingmeter-stick-genome-clinical-sequencing
15
N=1, Genome actionability
Volker: Intestinal surgery  XIAPCord blood
Beery twins: Cerebral palsy  SPRDiet 5HTP
Wartman: Leukemia  FLT3Sunitinib
Gilbert: Healthy BRCA Mas/Ovarectomy
Snyder#82: Healthy  GCKR, KCNJ11Diet, exercise
Smarr#74: Crohn’s  IL23R  Diet, probiotics
Bradfield: Healthy  CDH1  Gastrectomy
PGEd.org
10/18
SupercentenarianStudy.com
rare protective alleles
Calment 122.4
Breuning 114.6
Mortensen 115.7
Mirabella 110.6
Correlation → Cause → Cure/Prevention
Rare Protective alleles
•MSTN
•LRP5
•PCSK9
•CCR5
•FUT2
•APP
-/- Lean muscles
-/+ Extra-strong bones
-/+ Lower coronary disease
-/- HIV-resistant
-/- Stomach flu resistant
-/+ Alzheimer’s
blog.personalgenomes.org
<0.001%
0.001-8%
3, 0.06%
~0, 1%
20%
0.4%
18
18
N=1, Cause  Cure
"Long-Term Control of HIV by
CCR5 Δ32/Δ32 Stem-Cell
Transplantation" 2009
New England J Medicine
2007 Leukemia & AIDS:
Timothy Ray Brown
2012: Sangamo Phase 2 clinical trial
19
19
Cas9 CRISPR
RNA-guided human genome engineering. Science in press
Mali, Yang, Esvelt, Aach, Guell, DiCarlo, Norville, Church
Brain Activity
Map
(BAM+PGP)
Genes
Environments
Traits
PersonalGenomes.org
Biswal etal PNAS 2010
21
21
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 Dudley
Assistant Professor of Genetics and
Genomic Sciences and Director of
Biomedical Informatics
Mount Sinai
School of
Medicine
Webinar Sponsors