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What’s Next in Next-Generation Sequencing?

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.

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What’s Next in Next-Generation Sequencing?

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  1. What’s Next in Next-Generation Sequencing? Webinar Sponsors

  2. 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 1977 3D-RNA Rich, et al. Klug, et al. Kim, et al.

  3. From $3 billion to an affordable genomeWhen? Optimistic’ exponentialextrapolation DNA sequencing & Moore's law 1.5x/yr for electronics 6 decades

  4. From $3 billion to an affordable genomeWhen?6 years Earlyarrival DNA sequencing & Moore's law 1.5x/yr for electronics

  5. Sequencing via Imaging. Clinical AccuracyHaplotype phase: 2 mutations in cis vs trans 1.43 MbpLFR 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.

  6. Nanopore : Polymervs Monomer vsNanoTag 1988-1995: Church, Deamer, Branton, Baldarelli, Kasianowicz. 2012: Oxford & Genia 2009 Clarke, Bayley, et al 2010 Derrington, Gundlach, et al 2012Cherf, Akeson, et al GG AAA TTT CCC 5′ GCAACAGAGCCAGC CCC GCAACAGAGCCAGC AAA GCAACAGAGCCAGC CCC GCAACAGAGCCAGC TTT GCAACAGAGCCAGC GGA A15 3′. 6

  7. Nanopore: Polymer vs Monomer vsNanoTag “accuracy better than 1 in 5x108 events” A C GT 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

  10. RNA Lee, Terry, Daugharty, Turczyk, Scheiman, Yang, Li, Nick Conway; Collaborators --- Kun Zhang, Angela de Pace lab, FengZhang, Nilsson Probe extension with aminoallyl dUTP Probe immobilization using BS(PEG)9 pm pm •••••••••••••••••••••••••• Cy pm aU U aU Nu DNA ISH probe Nu Cy 5’ phos nm ISH probe trimming to prevent self-circularization aU aU USER cleavage ssDNA intramolecular circularization RCA primer hybridization RCA primer aU aU RCA with aminoallyl dUTP Amplicon immobilization using BS(PEG)9 Phi29 RCA Sequencing-by-ligation (1 z-slice shown) 6 base sequencing (1 z-slice shown) A G C T aU aU Fluorescent probe hybridization or sequencing

  11. Human fibroblast All RNAs GAPDH

  12. How can we improve genome interpretation?How we can make human omics data and cells shareable world-wide?

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

  14. GenomesEnvironmentsTraits (GET) 1660 0431 1846 1070 1730 1677 1687 1731 1833 1781 • World’s only open access data sets • Consented for re-IDentification • 100% onExam – 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 http://www.genomeweb.com/sequencing/nist-consortium-embarks-developing-meter-stick-genome-clinical-sequencing

  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 PGEd.org 10/18

  17. SupercentenarianStudy.comrare 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 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

  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

  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)GenesEnvironments TraitsPersonalGenomes.org Biswal etal PNAS 2010 21

  22. What’s Next in Next-Generation Sequencing? Webinar Sponsors

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