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 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 2. 60 yr. NGS. Epig, PersMed. Q&A after 3 talks. 1977 3D-RNA Rich, et al. Klug, et al. Kim, et al.

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.

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.

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?

Nanopore : Polymer vs Monomer vs NanoTag 1988-1995: 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

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

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

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.

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 800-1000nm 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.

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

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 2. 60 yr. NGS. Epig, PersMed. Q&A after 3 talks.

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 .

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

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  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

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 -/- 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 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 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