1. Carbon meets Silicon (& the $1000 human genome) Oct 9, 2002 HBS

2. gggatttagctcagtt gggagagcgccagact gaa gat ttg gag gtcctgtgttcgatcc acagaattcgcacca Post- 300 genomes & 3D structures 6

3. Commericial Advisory Roles & Technology-transfer Genome Pharmaceuticals 98-02 Caliper Technologies 94-02 CodonCode 96-02 GenProfileAG 97-02 Gendaq 00-1 EngeneOS 00-2 BeyondGenomics 00-2 Newcogen & Flagship 00-2 Longenity 01-2 Xeotron 01-02 Genomatica 01-2 Genome Therapeutics 89-94; Biogen 84-5 Tecan/Gamera 98-00 FamilyGenetix 00-1; This page was last updated 17-May-2002 by GMC. Biorad-Sadtler 79-81 Affymetrix 90-02 Millipore 89-90 Lynx 00-02 Pyrosequencing 01-2 Bruker Daltonics 93-7 Mosaic Technologies 93-01 Agilent 01-2 Aventis ‘98-01 MJ Research Inc. 86-02 Hamilton Co. 86-90 Intelligent Automation 92-6 Eli Lilly 98 Dupont 82-4

4. Famous human mutations PKU (preventable mental retardation) HbS (Malaria resistance) ApoE4 (dementia resistance) CCR5  32 (HIV resistance)

5. Pharmacogenomics Gene/Enzyme Drug Quantitative effect Examples of clinically relevant genetic polymorphisms influencing drug metabolism and effects. Additional data

6. 2-Oct-2002 Boston GSAC Panel Discussion "The Future of Sequencing Technology: Advancing Toward the $1,000 Genome" Moderators: J. Craig Venter, Ph.D., The Center for Advancement of Genomics Gerald Rubin, Ph.D., Howard Hughes Medical Institute Speakers: George Church, Ph.D., Harvard University Eugene Chen, Ph.D., US Genomics Tony Smith, Ph.D., Solexa Trevor Hawkins, Ph.D., Amersham Biosciences Corporation Susan Hardin, Ph.D., VisiGen Biotechnologies, Inc. Michael P. Weiner, 454 Corporation Daniel H. Densham, Mobious Genomics, Ltd

7. The impact of new technologies Digital computers & Networks 1968-93 WWW 1993-94 Recombinant DNA 1976-1986 Genome Project 1985-2002 Stem cells 1983-2002 Nanotechnology 1984-2002

8. Bionano-machines Types of biomodels. Discrete, e.g. conversion stoichiometry Rates/probabilities of interactions Modules vs “extensively coupled networks” Maniatis & Reed Nature 416, 499 - 506 (2002)

9. Steeper than exponential growth http://www.faughnan.com/poverty.html http://www.kurzweilai.net/meme/frame.html?main=/articles/art0184.html Moore's law of ICs 1965

10. How to do single DNA molecule manipulations?

11. Important alleles occur in “noncoding” non-conserved regions Lesch KP, et al Science 274:1527-31 Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region Piedrafita FJ, et al. JBC 271: 14412 Alu repeat SNP near the human Myeloperoxidase gene: “severalfold less transcriptional activity” "-463 G creates a stronger SP1 binding site... overrepresented in acute promyelocytic leukemia"

12. The issue is not speed, but hidden costs (e.g. accuracy & integration) Sub-microliter scale: 1  m = femtoliter ( 10 -15 ) Instruments <$100K per CPU. Why low-cost, high quality sequencing? & how much? Human genotypes 10 19 bp Immune B&T cell receptor spectra 10 10 bp (per year) Environment & pathogen monitoring ? RNA splicing in situ : 10 12 bits/mm 3 Compact storage 10 5 now to 10 17 bits/ mm 3 with DNA & How ?

13. Projected costs greatly affect our priorities bp/$ $/genome Method 1977 0.1 30B manual (pBR322) 1985 1 3B HGP goal 2002 10 300M de novo high-quality sequencing 2002 300 10M dd-polyphred raw-reseq 2002 2K 2M Perlegen, Lynx 2002 3M 1K per diploid? de novo? This session! 2002 10 13.0003 other data types (e.g. video)

14. New sequencing approaches in commercial R&D Method liter/bp LengthError Test-set $/device bp/hr Capil  fluidics e-6600 <0.1% 1e11 350k 80k ABI, Amersham, GenoMEMS, Caliper*, RTS* SeqByHyb e-12  1 <5% 1e9 200k 1M Perlegen-Affymetrix*, Xeotron* Mass Spectrometry Sequenom, Bruker* Single molecule>e-24 >>40? >80 30k-1M 180k Pore(Agilent*) Fluor(USGenomics, Solexa) FRET(VisiGen,Mobious) In vitro DNA-Amplification (e.g. Polonies) -- Multiplex cycles: Lynx*e-15 20<3% 1e7 ? 1M Pyroseq.* e-6>40<1% 1e6 100k 5k CisTran* e-13  1M? ParAllele, 454, RTS* *GMC has a potential financial interest (or Harvard license)

15. $1K per diploid human sequence Input: buccal cells, blood, or forensic samples. Output: prioritized list of deviant bps (e.g. non-conservative). Raw data rate: 16 pixels/bp, 1Mpixel per 6sec/CPU = 24 CPU days. Amortization: 5 yr for camera/CPU/transport @ $50K total = $200 per 10 11 bp Overhead: $200 /sq ft/yr * 40 sq.ft (400 cu.ft) = $40 Reagents: At 20  m per (5  m) polony and 40 bp reads means 10000 cm 2 area, 800 ml of fluor dNTP, $100/mg = $40 5 ml PCR reactions = $200 Disposables: 500 slides = $50 Electricity: 2 kwatts 24hr*24days* 0.13$/kwatt-hr = $150 Labor for repair: 10% of instrument cost = $10 Labor for operation: Slide PCR, slide dips, scans, etc. = $20 R&D: Initially NIH grants (i.e. 0% of this unbalanced budget). Total: per genome $710

16. Long-range continuity inspired by DNA-Fiber Fluorescent In Situ Hybridization 300 kb = 100 microns http://allserv.rug.ac.be/~fspelema/neubla/content/images_r.htm

17. Polony amplification & sequencing

18. Human DNA: Cystic Fibrosis CFTR gene 45 kbp Rob Mitra Vincent Butty Jay Shendure Ben Williams David Housman Hitomi Hutzell

19. A A A A A A B B B B B B A Single Molecule (library or natural A,B tags) B B A A Primer is Extended by Polymerase B A B A Polymerase colony (polony) In situ amplification (PCR, RCA, etc.) Primer A has 5 immobilizing Acrydite Mitra & Church Nucleic Acids Res. 27: e34

20. 1. Remove 1 strand of DNA. 2. Hybridize Universal Primer. 3. Add Red (Cy3) dTTP. BB 35 A G T.. T 4. Wash; Scan Red Channel BB 35 G C G.. Sequence polonies by sequential, fluorescent single-base extensions

21. 5. Add Green (FITC) dCTP 6. Wash; Scan Green Channel BB 35 A G T. T C BB 35 G C G.. C Sequence polonies by sequential, fluorescent single-base extensions

22. Base added: (C) A G T (C) (A) G (T) C (A) (G) T C A 3 TCACGAGT AGTGCTCA Sequencing multiple polonies Mitra &Shendure Alignment precision 0.4 pixel

23. Polony exclusion principle & Single pixel sequences Mitra & Shendure

24. Inexpensive, off-the-shelf equipment MJR in situ cycler Histology slide rack Microarray scanner

25. Polony in situ Sequencing Summary Integrated!: (purify), amplify, sequence, (separate) Femtoliter (1  m) scale Off-the-shelf equipment Chromosome haplotyping & RNA splice-typing In situ tissue compatible

26. Types of phenotypic effects of mutations PKU Trisomy 21 HbS