Analysis

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Analysis Synthesis of Omes
DOE Wed 3-Nov-2004 1130 AM
Thanks to Agencourt, Ambergen, Atactic,
BeyondGenomics, Caliper, Genomatica, Genovoxx,
Helicos, MJR, NEN, Nimblegen, ThermoFinnigan,
Xeotron/Invitrogen For more info see
arep.med.harvard.edu
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Systems Biology Loop
Synthetic Biology Tools
Metabolic optimality
Models
Experimental designs
(Systematic) Data
Flux Competitive growth
DNA RNA Polony-Seq
Syntheses Perturbations
Proteasome targeting Genome engineering
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

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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

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Motif Co-occurrence, comparative genomics, RNA
clusters, and/or ChIP2-location data
P 10-6 to 10-11
Genome Res. 14201208 Bulyk, McGuire,Masuda,Churc
h
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Synthetic testing of DNA motif combinations
1.3 2.4 (1.3 in
DargR) 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.
14201208
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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.

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(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. 284147
• Programming peptidomimetic syntheses by
  translating genetic codes designed de novo.
  Forster et al. 2003 PNAS 1006353
• High level cell-free expression specific
  labeling of integral membrane proteins. Klammt et
  al. 2004 Eur J Biochem 271568
• Cell-free translation reconstituted with purified
  components. Shimizu et al. 2001 Nat
  Biotechnol. 19751-5.

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in vitro genetic codes
5'
Second base
3'
A
C
U
A
U
C
A
C
mS
yU
U
G
eU
80 average yield per unnatural coupling.
bK biotinyllysine , mS Omethylserine
eU2-amino-4-pentenoic acid yU
2-amino-4-pentynoic acid
Forster, et al. (2003) PNAS 1006353-7
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Mirror world enzyme, parasite,
predator resistance access 2n 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
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Oligos for 150 776 synthetic genes(for E.coli
minigenome M.mobile whole genome respectively)
Forster Church
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Up to 760K Oligos/Chip18 Mbp for 700 raw
(6-18K genes)

• lt1K 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
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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 gt
ss-70-mer (chip)
gt ds-90-mer

gt ds-50-mer
20-mer PCR primers with restriction sites at the
50mer junctions
Tian, Gong, Sheng , Zhou, Gulari, Gao, Church
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Improve DNA Synthesis Accuracyvia mismatch
selection
Other mismatch methods MutS (H,L)
Tian Church
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Genome assembly
50 75 125 225 425 825
1002(n-1)
Moving forward 1. Tandem, inverted and
dispersed repeats (hierarchical assembly,
size-selection and/or scaffolding) 2. Reduce
mutations (goal lt1e-6 errors) to reduce of
intermediates 3. gt30 kbp homologous (Nick
Reppas) 4. Phage integrase site-specific
recombination, also for counters. Stemmer et al.
1995. Gene 16449-53Mullis 1986 CSHSQB.
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All 30S-Ribosomal-protein DNAs(codon
re-optimized)
1.7 kb
0.3 kb
Tian, Gong, Sheng , Zhou, Gulari, Gao, Church
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Improving synthesis accuracy 9-fold
Method Total bp Clones Trans-ition Trans-version Deletion Addition Bp/error
Hyb selection, PCR 23641 9 7 3 5 2 1391
Gel selection, PCR 24546 35 28 12 11 3 455
No selection, ligation PCR 6093 25 6 6 22 4 160
No selection, PCR 9243 21 25 13 19 1 159
Tian Church
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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.
Western blot based on His-tags
Tian Church
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Systems Biology Loop
Synthetic Biology Tools
Metabolic optimality
Models
Experimental designs
(Systematic) Data
Flux Competitive growth
DNA RNA Polony-Seq
Syntheses Perturbations
Proteasome targeting Genome engineering
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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 genotypephenotype
  associations
• Cell-lineage during development
• Phylogenetic footprinting, biodiversity

Shendure et al. 2004 Nature Rev Gen 5, 335.
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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)
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Single bacterial chromosome amplification
Ratio to unamplified hybridization along
the chromosome of Escherichia
Prochlorococus on Affymetrix chips.
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Convergence on non-electrophorectic tag
sequencing methods?

• Tag gt400 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 gt Agencourt "Bead Polonies"
• 2 Kawashima'88, Adams'97 gt Lynx/Solexa
  "Clusters"

http//arep.med.harvard.edu/Polonator/Plone.htm
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Polony Fluorescent In Situ Sequencing Libraries
1 to 100kb Genomic
2x20bp after MmeI (BceAI, AcuI)
L R M
M
Sequencing primers
PCR bead
Greg Porreca Abraham Rosenbaum
Dressman et al PNAS 2003 emulsion
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Cleavable dNTP-Fluorophore ( terminators)
Reduce or photo- cleave
Mitra,RD, Shendure,J, Olejnik,J, Olejnik,EK, and
Church,GM (2003) Fluorescent in situ Sequencing
on Polymerase Colonies. Analyt. Biochem. 32055-65
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Polony-FISSeq up to 2 billion beads/slide
0.5 of full gel area
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Polony-FISSeq up to 2 billion beads/slide Cy5
primer (570nm) Cy3 dNTP (666nm)
Jay Shendure
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Polony FISSeq Stats

• of bases sequenced (total) 23,703,953
• bases sequenced (unique) 73
• Avg fold coverage 324,711 X
• Pixels used per bead (analysis) 3.6
• Read Length per primer 14-15 bp
• Insertions 0.5
• Deletions 0.7
• Substitutions (raw) 4e-5
• Throughput 360,000 bp/min

Current capillary sequencing 1400 bp/min (600X
speed/cost ratio, 5K/1X) (This may omit PCR ,
homopolymer, context errors)
Shendure
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Systems Biology Loop
Synthetic Biology Tools
Metabolic optimality
Models
Experimental designs
(Systematic) Data
Flux Competitive growth
DNA RNA Polony-Seq
Syntheses Perturbations
Proteasome targeting Genome engineering
30
.
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High accuracy special case homopolymers (e.g.
AAA, CC, etc.)

• Use "compressed" tags , ACG ACCGACCCG
• Quantitate incorporation
• Reversible terminators
• "Wobble sequencing"
• All of these work.
• Maintenance of amplification fidelity using
  linear amplification from initial genomic
  fragment

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"Wobble sequencing" for homopolymers

• 6 positions 16 primers 4 dNTPs gt 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)