1. Thanks to:.gov ||.edu ||.org ||.com || Read = = = = = = = = I/O = = = = = = = Write Little Data: NextGen Info Storage in DNA 3:55-4:50 PM CBA 7-Mar-2013 12:12-12:21 PM Azco ArmRev.org Oppenheimer Foundation Gen9 LSRF NHGRI NIGMS

2. Next-generation Digital Information Storage in DNA 1.Read/write DNA 10-fold/yr: Next = FISSeq 2. E.coli & human genome engineering 3. DNA data archiving 4. Brain Activity Map I/O: Polymerase Biologically Inspired Engineering

3. 3 From $3 billion to an affordable genome When? DNA sequencing & Moore's law 1.5x/yr for electronics ‘Optimistic’ exponential extrapolation 6 decades

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

5. 5 Fluorescent in situ Sequencing (FISSEQ) 60 cycles x 4 colors Lee, Yang, Terry, Nilsson, Church et al. Single base differences

6. Next-generation Digital Information Storage in DNA 1.Read/write DNA 10-fold/yr: Next = FISSeq 2. E.coli & human genome engineering 3. DNA data archiving 4. Brain Activity Map I/O: Polymerase Biologically Inspired Engineering

7. 7 113 kbp DNA 151 genes Pure translation: Forster & Church MSB ’05 GenomeRes.’06 Shimizu, Ueda ’01 Smaller. Higher speed & accuracy requires a few extra genes (E.coli 20 min. doubling) Reconstituted ribosomes: Jewett & Church

8. First Genomically Recoded Organism (GRO) 3 New Biological Functions Lajoie MJ, Rovner AJ, Goodman DB, Aerni HR, Haimovich AD, Mercer JA, Wang HH, Carr PA, Mosberg JA, Rohland N, Schultz PG, Jacobson JM, Rinehart J, Church GM, Isaacs FI 4.7 Mbp Metabolic dependence Safety Virus resistance Safety New Amino Acids

9. 9 Multiplex Assembly Genome Engineering (MAGE) #1: ss-90mer x ds-Circle #2: ss-Mb x ds-Circle conjugation Costantino &Court PNAS’03 Wang et al., Nature '09 MAGE 200 kbp BAC CAGE Isaacs et al., Science ‘11 #3: CRISPR #4: 10 Mbp Microcell-Mediated Chromosome Transfer (MMCT)

10. 10 Targeted Gene Therapy ZnF nuclease CCR5 double null in T-cells Sangamo Phase 2 clinical trial (AIDS treatment) Next: CRISPR RNA guided Nucleases 2007 Leukemia & AIDS: Timothy Ray Brown

11. Next-generation Digital Information Storage in DNA 1.Read/write DNA 10-fold/yr: Next = FISSeq 2. E.coli & human genome engineering 3. DNA data archiving 4. Brain Activity Map I/O: Polymerase Biologically Inspired Engineering

12. DNA+mAb 1977: 3- & 4-way DNA. 2009: caDNAno.org 2012: Douglas, Bachelet, Church. Science Nanorobot: Sensors, Logic, Actuators

13. Amplify pools with flanking universal primers Paths to error correction 1.Hyb-Select: Tian et al. 2004 Nature 2. MutS: Carr & Jacobson 2004 NAR 3. MutHLS: Smith & Modrich 1997 PNAS 4. Endo/Exonuclease : Bang Nat Meth. 2008 5. Errase 6. Sequencing Next-Gen Synthesis 12K CustomArray Electrolytic 55K Agilent Ink-jet 80K LCSci/Mycroarray Photo-Acid 6.3M Roche Photo 5’ protection ($0.2K-$13K / chip) 13

14. Archival DNA. Zettabyte/kg Images  Camera  Computer  Inkjet  DNA  Polymerase Text printer copying Images  RNA Polymerase DMD + Nitrobenzyl

15. DNA-video 2013 15 Digital Inkjet 2012 2012 Church, et al 2013 Goldman et al. 1986 Davis, et al. Kosuri

16. Next-generation Digital Information Storage in DNA 1.Read/write DNA 8-fold/yr 2.Fluorescent In Situ Sequencing (FISSeq) 3.Nanorobots & DNA data archiving 4.Brain Activity Map I/O: Polymerase Biologically Inspired Engineering

17. 17 Brain Activity Map (BAM) Sep 2011: Kavli, Gatsby, Allen: Chun Imaging: Greenspan, Yuste, Zhaung, Reid Optical fibers: Roukes, Boyden, Scherer Nano: Alivisatos, Park, Weiss, McEuen Stimulation: Donoghue, Deisseroth Computation: Kording, Sejnowski Polymerase: Zamft, Marblestone, Schmidt, Tyo, Martín-Alarcón, Marcus

18. 18 Epilepsy & Images via Electrode Wires Quiroga et al. 2005 Nature. Invariant visual representation by single neurons in the human brain (left posterior hippocampus)

19. Number of Simultaneously Recorded Neurons Konrad Kording Scaling of Brain Mapping Technologies

20. 20 IC energy efficiency 1.5x /yr economist.com/node/2153 1350 Polymerase 7e25 Op/kWh

21. DNA Pol Ions to DNA Light to DNA ATP to DNA

22. DNA Pol Ions to DNA Zamft, Marblestone et al. PLoS One 2012 Dpo4 Pol

23. .

24. .

25. 25 Brain Activity Map I/O Why? Blindness, deafness, stroke, traumatic brain injuries, spinal cord injuries, Parkinson’s, chronic pain, depression, locked-in syndrome, dystonia, essential tremor, epilepsy, neurodegenerative diseases. Alivisatos, Chun, Church, Donoghue, Greenspan, Roukes, Yuste; The Brain Activity Map Project and the Challenge of Functional Connectomics. Neuron 2012

26. 26 Brain Activity Map I/O Miesenböck Science 2009 Ju Lu, Harvard University

27. 27 Wireless molecular I/O. 100M Neural cells

28. Next-generation Digital Information Storage in DNA 1.Read/write DNA 8-fold/yr 2.Fluorescent In Situ Sequencing (FISSeq) 3.Nanorobots & DNA data archiving 4.Brain Activity Map I/O: Polymerase Biologically Inspired Engineering

29. . Zorzos et al 2012 Optics Letters

30. 42nm spacing fluorescent barcodes. Nature Chem 2012 Lin, Jungmann, Leifer, Li, Levner, Church, Shih, Yin

31. Jay Lee Evan Daugharthy Reza Kalhor Jonathan Scheiman Michael Sismour Joyce Yang John Aach Kun Zhang Yoav Mayshar Polonator & Advanced Technology Team: Nicholas Conway Daniel Levner Chao Li Richard Terry Brian Turczyk Frederick Vigneault Fluorescent In situ Sequencing FISSEQ RNA & DNA

32. Generic Health Advice Exercise Drink your milk Eat your grains & beans & iron & meat Get more rest

33. UNLESS … ExerciseHCM (cardiac arrest) Drink your milkMCM6 (gastric distress) Eat your grainsHLA-DQ2 (diarrhoea) & beansG6PD (red cell lysis) & ironHFE (Liver damage) & meatLDLR (clogged arteries) Get more restHLA-DR2 (excessive sleep)

34. 34 Most new Sequencing = single-molecule imaging 1. Danaher MA 2. Roche-454 CT 3. AB-SOLiD MA 4. Illumina UK,CA 5. CGI/BGI CA 6. Helicos MA 7. Pacific Bio CA 8. IBS/Qiagen MA 9. Ion Torrent CT 17. LightSpeed CA 10. Genapsys CA 11. Electronic Biosci CA 12. Nabsys RI 13. OxfordNanopore UK 14. Genia CA 15. IBM-Roche NY 16. NobleGen MA 18. GnuBio MA 19. Bionanomatrix PA 20. Halcyon CA 21. ZS Genetics NH 22. Electron Optica CA 23. Genizon BioSci QC 24. LaserGen TX 25. GE Global NY 26. Stratos Genomics WA 27. Reveo NY 28. Firebird FL 29. Zeiss MA 30. Lucigen WI 31. Adv. Liquid Logic NC 32. Caerus Molec Diag CA http://arep.med.harvard.edu/gmc/nexgen.html 33. Nanophotonics Biosci CA 34. Network Biosystems MA 35. SeiraD NM 36. Affymetrix CA 37. Population Gen Tech UK 38. AQI Sciences AZ 39. Base4innovation UK 40. Li-Cor NE 41. U.S. Genomics MA 42. Mobious Genomics UK 43. Visigen TX 44. Starlight CA

35. 35 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′.

36. Nanopore: Polymer vs Monomer vs NanoTag PEG-Labeled PP: Ju, Kasianowicz, et al. Scientific Reports 2012 “accuracy better than 1 in 5x10 8 events” A C G T Oxford, Genia