1. 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

2. 60 yr + 3D 1965 1D-RNA sequence 2013? Holley, Complete 1D genome?
Everett,
Madison,
Zamir
2013?
Complete 1D genome?
3D-genome?
3D-transcriptome?
1953
3D-generic DNA
Franklin,
Gosling,
Watson,
Crick,
Wilkins,
Stokes,
Wilson
18 min. Shutdown other programs. 2:15 PM ET login. 2:30 PM. GC:Open access. GW:Microbio. JD: Informatics. Screen yr.
NGS. Epig, PersMed. Q&A after 3 talks.
1977
3D-RNA
Rich, et al.
Klug, et al.
Kim, et al.

3. From $3 billion to an affordable genome When?
Optimistic’ exponential extrapolation
6 decades
From $3 billion to an affordable genome When?
DNA sequencing
& Moore's law
1.5x/yr for electronics
What makes this possible? Factors of 1.5-fold per yr just like computers until 2005 & since then factors of ten per yr. So called next generation sequencing.

4. From $3 billion to an affordable genome When? 6 years
Early arrival
From $3 billion to an affordable genome When? 6 years
DNA sequencing
& Moore's law
1.5x/yr for electronics
What makes this possible? Factors of 1.5-fold per yr just like computers until 2005 & since then factors of ten per yr. So called next generation sequencing.

5. Sequencing via Imaging
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.
How can each of these fail individually?

6. Nanopore : Polymer vs Monomer vs NanoTag
: Church, Deamer, Branton, Baldarelli, Kasianowicz.
2012: Oxford & Genia
2009 Clarke, Bayley, et al
2010 Derrington, Gundlach, et al
2012 Cherf, Akeson, et al
GG AAA
TTT CCC .
5′ GCAACAGAGCCAGC CCC
GCAACAGAGCCAGC AAA GCAACAGAGCCAGC CCC
GCAACAGAGCCAGC TTT GCAACAGAGCCAGC GG A A15 3′. 6 6

7. Nanopore: Polymer vs Monomer vs NanoTag
“accuracy better than
1 in 5x108 events” A C
G T
Some of the environmental components in red are genomic and epigenomics in green with costs dropping a million fold. Also medical records via Google Health
PEG-Labeled PP: Ju, Kasianowicz, et al.
Scientific Reports 2012

8. Fluorescent in situ Sequencing (FISSEQ) 60 cycles x 4 colors (3D omes)
Single base
differences
Lee, Yang, Terry, Nilsson, Church et al.

9. Danaher Polonator 2013: Automation & Image Processing
Working with the advanced technology team, we have also started to automate the data acquisition and processing aspects of the method. This is an image of the new polonator, which is a combination of liquid handling and imaging capabilities.

10. Lee, Terry, Daugharty, Turczyk, Scheiman, Yang, Li, Nick Conway; Collaborators --- Kun Zhang, Angela de Pace lab, Feng Zhang, Nilsson
DNA ISH probe
USER cleavage
Probe extension with aminoallyl dUTP
Probe immobilization using BS(PEG)9
•••••••••••••••••••••••••• aU
RNA
RCA primer
Phi29
RCA
ISH probe trimming to prevent self-circularization
RCA with aminoallyl dUTP
Amplicon immobilization using BS(PEG)9
Fluorescent probe hybridization or
sequencing
ssDNA intramolecular circularization
RCA primer hybridization A G C T
6 base sequencing
(1 z-slice shown)
Sequencing-by-ligation
5’ phos U Nu Cy pm nm
Slide one
A.RT primers (polydT, random hexamer or gene specific) are bound and extended briefly (the average size is unknown, but expected to be around 500-bp based on literature (larsson et al. Nature Method). We have found that the sweet spot for the efficient circualrization and amplication to be between 50 to 200 additional bases.) The cDNA is then immobilized using amine to NHS crossliker chemistry containing a 4 nm spacer (BSPEG9). The total RNA is digested and dephosphorylated, followed by uncapping of the bound primer. Target specific RCA primer is then used to amplify the cDNA sequence tags. The DNA rolonies are then cross linked into a semi-permanent 3D matrix that can be hybridized repeatly or sequenced in situ.
B. iPS cells are primed with random hexamers. Using 1000x scanning zoom confocal microscopy, one can visualize individual rolonies packing densly within the cell, counterstaining the nuclear and the plasma membrane, the condensed chromatins and vesicles. When no RT is done, we find very little amplicons. In iPS cells, the total transcriptome are sometimes exclusively cytoplasmic (first panel; also in the nucleolus) or highly enriched in the nucleus, esp. during the late G2 phase to metaphase.
C. These samples can be stripped of their fluoroscent primers and rehybridized without the loss of signal to noise ratio for more than 60 cycles.
After 12 cycles of sequencing by ligation, the computation spot tracking algorithm (Imaris) finds that the average drift of each spot is 500 nm (for nm rolonies) in x-y-z direction.
D. The total transcriptome amplicons can be stripped and only a specific set of amplicons can be sequenced using different sequencing primers (here, the sequencing primer expected to detect only 25% of the overal amplicons are used); all experiments are done on 63x scanning confocal.
E. Here you can see that the majority of the bright dots representing a sequence call align well over the duration of the sequencing cycle. This is one optical slice (250 nm thick) out of 19 slices, arranged vertically over time.

11. Human fibroblast All RNAs GAPDH
Here is one of our best results, where we have sequenced GAPDH within human fibroblasts. You can see that on the left random sequencing gives fluorescence in all four channels (one channel per base), while over 90% of GAPDH seqeuncing is in the blue channel – which is the expected base, with little off-target sequencing.
All RNAs
GAPDH

12. How can we improve genome interpretation
How can we improve genome interpretation? How we can make human omics data and cells shareable world-wide?
18 min. Shutdown other programs. 2:15 PM ET login. 2:30 PM. GC:Open access. GW:Microbio. JD: Informatics. Screen yr.
NGS. Epig, PersMed. Q&A after 3 talks.

13. Genomes + Environments = Traits
Nutrition
Chemicals
Immunome
Stem-cells
Epigenome (RNA,mC)
Cancer
PersonalGenomes.org
(US, Canada, UK, EU goals: 100,000 volunteers each)
Microbiome
Therapies
evidence.personalgenomes.org
TRAITS
(Phenome)
PERSONAL GENOME
3M alleles .

14. Genomes Environments Traits (GET)
0431
1070
1660
1677
1687
1833
1846
1731
1730 
1781
World’s only open access data sets
Consented for re-IDentification
100% on Exam – Educate first
Stem Cell Biobank

15. 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

16. 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
Nic,… Lukas, Allison
PGEd.org
10/18

17. SupercentenarianStudy.com rare protective alleles
Calment 122.4
Breuning 114.6
Mortensen 115.7
Mirabella 110.6

18. Correlation → Cause → Cure/Prevention Rare Protective alleles
MSTN -/- Lean muscles <0.001%
LRP5 -/+ Extra-strong bones %
PCSK9 -/+ Lower coronary disease , 0.06%
CCR5 -/- HIV-resistant ~0, 1%
FUT /- Stomach flu resistant %
APP /+ Alzheimer’s % .
blog.personalgenomes.org 18 18

19. 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

20. Cas9 CRISPR RNA-guided human genome engineering. Science in press
Mali, Yang, Esvelt, Aach, Guell, DiCarlo, Norville, Church

21. Brain Activity Map (BAM+PGP) Genes Environments Traits PersonalGenomes
Brain Activity Map (BAM+PGP) Genes Environments Traits PersonalGenomes.org
Biswal etal PNAS 2010 21 21

22. 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