Thanks to: DARPA & DOE-GtL Agencourt, Ambergen, Atactic, BeyondGenomics, Caliper, Genomatica, Genovoxx, Helicos, MJR, NEN, Nimblegen, Xeotron/Invitrogen For more info see: arep.med.harvard.edu 1-Feb :15-10 MITRE Low-Cost/High-Accuracy Microbial Genome Synthesis and Monitoring

Synthetic - homologous recombination testing of DNA motifs (1.3 in  argR) RNA Ratio (motif- to wild type) for each flanking gene Bulyk, McGuire,Masuda,Church Genome Res. 14:201–208

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. Protein design, vaccines, solubility screens Utility of molecular biology DNA -- RNA -- Protein in vitro "kits" (e.g. PCR -- T7 -- Roche) Toward these goals design a chassis: 115 kbp genome. 150 genes. Nearly all 3D structures known. Comprehensive functional data.

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

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: % average yield per unnatural coupling. eU = 2-amino-4-pentenoic acid yU = 2-amino-4-pentynoic acid mS = O-methylserine gS = O-GlcNAc–serine bK = biotinyl-lysine

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

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

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 => 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

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

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. 15kb to 5Mb by homologous recombination (Nick Reppas) 4. Phage integrase site-specific recombination, also for counters. Stemmer et al Gene 164:49-53;Mullis 1986 CSHSQB … 100*2^(n-1)

All 30S-Ribosomal-protein DNAs (codon re-optimized) Tian, Gong, Sheng, Zhou, Gulari, Gao, Church 1.7 kb 0.3 kb s19 0.3kb Nimblegen 95K chip Atactic <4K chip

Improving synthesis accuracy Method Bp/error Chip assembly only 160 Hybridization-selection 1,400 MutS-gel-shift 10,000 MutHLS cleavage 30,000 (10X better than PCR) Tian & Church 2004 Carr & Jacobson 2004 Smith & Modrich 1997

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

Safety Proposals Church, G.M. A synthetic biohazard non-proliferation proposal. (2004) ( Monitor oligo synthesis via expansion of Controlled substances, Select Agents, &/or Recombinant DNA 2. Computational tools for the above 3. System modeling checks for synthetic biology projects 4. Multi-auxotroph, novel genetic code for the host genome, prevents functional transfer of DNA to other cells.

Why sequence? Synthetic biology & laboratory selections Pathogen "weather map", biowarfare sensors Cancer: mutation sets for individual clones, loss-of-heterozygosity RNA splicing & chromatin modification patterns. 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 Nature Rev Gen 5, 335.

Personal genomics & cancer therapy Mutations G719S, L858R, Del746ELREA in red. EGFR Mutations in lung cancer: correlation with clinical response to gefitinib [Iressa] therapy. Paez, … Meyerson (Apr 2004) Science 304: 1497 Lynch … Haber, N Engl J Med. (Apr 2004) 350:2129. Pao.. Mardis,Wilson,Varmus H, PNAS (Aug 2004) 101: Dulbecco R. (1986) A turning point in cancer research: sequencing the human genome. Science 231:

Why 'single molecule' sequencing? (1) Single-cells: Preimplantation (PGD), uncultivatable (2) Co-occurrence on a molecule, complex, cell RNA splice-forms & DNA haplotypes (3) Cost: $1K-100K "personal genomes" (4) Precision: Counting 10 9 RNA tags (to reduce variance) (~5e5 RNAs per human cell) Fixed 5e3 5e4 5e6 5e9 (goal) costs EST SAGE MPSS Polony-FISSeq (polymerase colony)

CD44 Exon Combinatorics (Zhu & Shendure) Alternatively Spliced Cell Adhesion Molecule Specific variable exons are up-or-down-regulated in various cancers (>2000 papers) v6 & v7 enable direct binding to chondroitin sulfate, heparin… Zhu,J, et al. Science. 301:836-8.

Zhu J, Shendure J, Mitra RD, Church GM. Science 301: Single molecule profiling of alternative pre-mRNA splicing. Eph4 = murine mammary epithelial cell line Eph4bDD = stable transfection of Eph4 with MEK-1 (tumorigenic) CD44 RNA isoforms

Chromosome-wide haplotyping IL : A 60-Mb CD : A /T Human Chr. 7 A..A A..T Mb

Convergence on non-electrophorectic tag-sequencing methods? Tag > bp (2-ends) EST SAGE MPSS 454 Polony-Seq Ronaghi 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"

Bead Polony Sequencing Pipeline In vitro libraries via paired tag manipulation Bead polonies via emulsion PCR [Dre03] Monolayered immobilization in acrylamide Enrichment of amplified beads SOFTWARE Images → Tag Sequences Tag Sequences → Genome FISSEQ or “wobble” sequencing Epifluorescence Scope with Integrated Flow Cell

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

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

Polony- FISSeq : up to 2 billion beads/slide Cy5 primer (570nm) ; Cy3 dNTP (666nm) Jay Shendure Self Organizing Monolayer

# 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

High accuracy special case: homopolymers (e.g. AAA, CC, etc.) Use "compressed" tags, ACG = ACCG=ACCCG Quantitate incorporation Reversible terminators FRET between adjacent 3' bases Wobble sequencing All five of these work. Maintenance of amplification fidelity using linear amplification from initial genomic fragment

Degenerate (aka “wobble) sequencing “single tipped” vs “double tipped” length of anchoring sequence natural vs. universal nucleotides (i.e. deoxyinosine) single fluor vs. four-color fluor mixtures of dNTPs for extensions Sequenase vs Klenow vs BST Exonuclease stripping vs heat stripping CTAGCGAGCTAGNNNNNNNNA CTAGCGAGCTAGNNNNNNNNG CTAGCGAGCTAGNNNNNNNNC CTAGCGAGCTAGNNNNNNNNT anchordegenerate “tip”

Wobble vs Simple base-extension 1/4 vs 2.5/4 base/cycle >8 vs base reads 3e-3 vs 4e-5 non-homopolymer errors 3e-3 vs 1e-1 homopolymer errors 40' per cycle, 60 hr per 20 cycles

Sequencing single molecules Ecosystem studies need single-cell amplification because of multiple chromosomes (& RNAs) per cell. Many cells are hard to grow. Microbes exchange genome subsets. (Even an 80% genome coverage is better than 100 kb BACs) Many input molecules required to sequence one molecule. vs. one molecule sufficient to sequence via many copies of it.

Single cell sequencing  29 real-time amplification No template control Affymetrix quantitation of independent amplifications

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