1. Thanks to: Agencourt, Ambergen, Atactic, BeyondGenomics, Caliper, Genomatica, Genovoxx, Helicos, MJR, NEN, Nimblegen, ThermoFinnigan, Xeotron/Invitrogen For more info see: arep.med.harvard.edu DOE Wed 3-Nov-2004 11:30 AM Analysis & Synthesis of Omes

2. Systems Biology Loop Syntheses & Perturbations Models Experimental designs (Systematic) Data Proteasome targeting Genome engineering Metabolic optimality Flux & Competitive growth DNA & RNA Polony-Seq Synthetic Biology Tools

3. DOE Synthetic Genomes: Why? Cheaper/faster "standard biology", hypothesis testing Systems Biology: Multiple simultaneous tests Viruses: Aid strain transfer; generate variants, new haplotypes Anti-viral vaccines and therapeutics (including variants) In vitro: Make products toxic in E.coli. Microbes: Interspecific hybrids (e.g. codon usage) Structural biology: variants Rapid vaccine response to engineered bioterrorism. Cell-mediated immunity + humoral. Fix mismatch between genome analysis & synthesis

4. DOE Synthetic Genomes: Why? In vitro Microbial & Human Antimutators Artificial ecosystems (laboratory scales) Energy aiding pathway improvement Instrustrial production: Enzymes, SingleCellProtein, Protein- drugs Remediation: Hybrid genomes (opt. codons), combinatorial pathway (Maxygen & Diversa). Xylose & Oil Pharmaceuticals: Combinatorial syntheses Nano science Combinatorial syntheses, Complex nanosystems, more general nanoassembly (in reach of polymerases and ribosome-like factories) Health research: 10X faster results per current $ (cost/benefit) Hypothesize & test unknown gene combinations Synthetic standards (arrays, MS, quantitation, etc) Agriculture: salt, cold, drought, pest tolerant hybrid genomes

5. Motif Co-occurrence, comparative genomics, RNA clusters, and/or ChIP 2 -location data P= 10 -6 to 10 -11 Genome Res. 14:201–208 Bulyk, McGuire,Masuda,Church

6. Synthetic testing of DNA motif combinations 1.3 2.4 (1.3 in  argR) 1.1 1.3 0.7 2.5 0.2 1.4 1.4 3.5 RNA Ratio (motif- to wild type) for each flanking gene Bulyk, McGuire,Masuda,Church Genome Res. 14:201–208

7. Synthetic Genomes & Proteomes. Why? Test or engineer cis-DNA/RNA-elements Access to any protein (complex) including post-transcriptional modifications Affinity agents for the above. Mass spectrometry standards, protein design Utility of molecular biology DNA-RNA-Protein in vitro "kits" (e.g. PCR, SP6, Roche) Toward these goals design a chassis: 115 kbp genome. 150 genes. Nearly all 3D structures known. Comprehensive functional data.

8. (PURE) translation utility Removing tRNA-synthetases, translational release-factors, RNases & proteases Selection of scFvs specific for HBV DNA polymerase using ribosome display. Lee et al. 2004 J Immunol Methods. 284:147 Programming peptidomimetic syntheses by translating genetic codes designed de novo. Forster et al. 2003 PNAS 100:6353 High level cell-free expression & specific labeling of integral membrane proteins. Klammt et al. 2004 Eur J Biochem 271:568 Cell-free translation reconstituted with purified components. Shimizu et al. 2001 Nat Biotechnol. 19:751-5.

9. in vitro genetic codes 5' mS yU eU UGG UUG CAG AAC... GUU A 3' GAAACCAUG fMTNVE | | | 5' Second base 3' U A C C U mS yU eU A C U G A Forster, et al. (2003) PNAS 100:6353-7 80% average yield per unnatural coupling. bK = biotinyllysine, mS = Omethylserine eU=2-amino-4-pentenoic acid yU = 2-amino-4-pentynoic acid

10. Mirror world : enzyme, parasite, & predator resistance & access 2 n diastereomers (n chiral atoms) L-amino acids & D-ribose (rNTPs, dNTPs) Transition: EF-Tu, peptidyl transferase, DNA-ligase D-amino acids & L-ribose (rNTPs, dNTPs) Dedkova, et al. (2003) Enhanced D-amino acid incorporation into protein by modified ribosomes. J Am Chem Soc 125, 6616-7

11. Forster & Church Oligos for 150 & 776 synthetic genes (for E.coli minigenome & M.mobile whole genome respectively)

12. Up to 760K Oligos/Chip 18 Mbp for $700 raw (6-18K genes) <1K Oxamer Electrolytic acid/base 8K Atactic/Xeotron/Invitrogen Photo-Generated Acid Sheng, Zhou, Gulari, Gao (U.Houston) 24K Agilent Ink-jet standard reagents 48K Febit 100K Metrigen 380K Nimblegen Photolabile 5'protection Nuwaysir, Smith, Albert Tian, Gong, Church

13. Improve DNA Synthesis Cost Synthesis on chips in pools is 5000X less expensive per oligonucleotide, but amounts are low (1e6 molecules rather than usual 1e12) & bimolecular kinetics slow with square of concentration decrease!) Solution: Amplify the oligos then release them. 10 50 10 => ss-70-mer (chip) 20-mer PCR primers with restriction sites at the 50mer junctions Tian, Gong, Sheng, Zhou, Gulari, Gao, Church => ds-90-mer => ds-50-mer

14. Improve DNA Synthesis Accuracy via mismatch selection Tian & Church Other mismatch methods: MutS (&H,L)

15. Genome assembly Moving forward: 1. Tandem, inverted and dispersed repeats (hierarchical assembly, size-selection and/or scaffolding) 2. Reduce mutations (goal <1e-6 errors) to reduce # of intermediates 3. >30 kbp homologous (Nick Reppas) 4. Phage integrase site-specific recombination, also for counters. Stemmer et al. 1995. Gene 164:49-53;Mullis 1986 CSHSQB. 50 75 125 225 425 825 … 100*2^(n-1)

16. All 30S-Ribosomal-protein DNAs (codon re-optimized) Tian, Gong, Sheng, Zhou, Gulari, Gao, Church 1.7 kb 0.3 kb

17. Improving synthesis accuracy 9-fold Method Total bp # Clones Trans- ition Trans- versionDeletionAddition Bp/error Hyb selection, PCR2364197352 1391 Gel selection, PCR24546352812113 455 No selection, ligation +PCR60932566224 160 No selection, PCR9243212513191 159 Tian & Church

18. Extreme mRNA makeover for protein expression in vitro RS-2,4,5,6,9,10,12,13,15,16,17,and 21 detectable initially. RS-1, 3, 7, 8, 11, 14, 18, 19, 20 initially weak or undetectable. Solution: Iteratively resynthesize all mRNAs with less mRNA structure. Tian & Church Western blot based on His-tags

19. Systems Biology Loop Syntheses & Perturbations Models Experimental designs (Systematic) Data Proteasome targeting Genome engineering Metabolic optimality Flux & Competitive growth DNA & RNA Polony-Seq Synthetic Biology Tools

20. Why sequence? Cancer: mutation sets for individual clones, loss-of-heterozygosity Pathogen "weather map", biowarfare sensors RNA splicing & chromatin modification patterns. Synthetic biology & lab selections Antibodies or "aptamers" for any protein B & T-cell receptor diversity: Temporal profiling, clinical Preventative medicine & genotype–phenotype associations Cell-lineage during development Phylogenetic footprinting, biodiversity Shendure et al. 2004 Nature Rev Gen 5, 335.

21. Sequencing single molecules Ecosystem studies really need single-cell amplification because of multiple chromosomes (& RNAs) (Even an 80% genome coverage is better than 100 kb BACs)

22. Single bacterial chromosome amplification Ratio to unamplified hybridization along the chromosome of Escherichia & Prochlorococus on Affymetrix chips.

23. Convergence on non-electrophorectic tag sequencing methods? Tag >400 14-26 20 100 26 bp (2-ends) EST SAGE MPSS 454 Polony-Seq Single-molecule vs. amplified single molecule. Array vs. bead packing vs. random Rapid scans vs. long scans (chemically limited, 454) Number of immobilized primers: 0: Chetverin'97 "Molecular Colonies" 1: Mitra'99 > Agencourt "Bead Polonies" 2: Kawashima'88, Adams'97 > Lynx/Solexa: "Clusters" http://arep.med.harvard.edu/Polonator/Plone.htm

24. Polony Fluorescent In Situ Sequencing Libraries Greg Porreca Abraham Rosenbaum 1 to 100kb Genomic M L R M PCR bead Sequencing primers Selector bead 2x20bp after MmeI ( BceAI, AcuI) Dressman et al PNAS 2003 emulsion

25. Cleavable dNTP-Fluorophore (& terminators) Mitra,RD, Shendure,J, Olejnik,J, Olejnik,EK, and Church,GM (2003) Fluorescent in situ Sequencing on Polymerase Colonies. Analyt. Biochem. 320:55-65 Reduce or photo- cleave

26. 0.5% of full gel area Polony-FISSeq: up to 2 billion beads/slide

27. Cy5 primer (570nm) ; Cy3 dNTP (666nm) Jay Shendure

28. # of bases sequenced (total)23,703,953 # bases sequenced (unique)73 Avg fold coverage324,711 X Pixels used per bead (analysis)~3.6 Read Length per primer14-15 bp Insertions 0.5% Deletions 0.7% Substitutions (raw) 4e-5 Throughput:360,000 bp/min Polony FISSeq Stats Current capillary sequencing 1400 bp/min (600X speed/cost ratio, ~$5K/1X) (This may omit: PCR, homopolymer, context errors) Shendure

29. Systems Biology Loop Syntheses & Perturbations Models Experimental designs (Systematic) Data Proteasome targeting Genome engineering Metabolic optimality Flux & Competitive growth DNA & RNA Polony-Seq Synthetic Biology Tools

30. .

31. High accuracy special case: homopolymers (e.g. AAA, CC, etc.) Use "compressed" tags, ACG = ACCG=ACCCG Quantitate incorporation Reversible terminators "Wobble sequencing" All of these work. Maintenance of amplification fidelity using linear amplification from initial genomic fragment

32. "Wobble sequencing" for homopolymers 6 positions * 16 primers * 4 dNTPs => 13 bp (paired ends) CCTCATTCTCT AA + dATP (then C, …) CCTCATTCTCT AC + dATP (then C, …)... CCTCATTCTCTnnAA + dATP (then C, …)... CCTCATTCTCTnnNNnnNNnnTT + dATP (then C, …) 4.5/64 bp/cycle (for wobble sequencing) vs. 2.5/4 bp/cycle (for simple sequential base-extension)