Integrating Optoelectronic, Fluidic

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Integrating Optoelectronic, Fluidic Biochemical
Programming For Open-Source Personal Genomics
Synthetic Biology
George Church Thu 24-May-2007 MIT E15 Coding
and Computation in Microfluidics
Thanks to
AppliedBiosystems, Helicos, Roche454, Illumina,
CGI, IBS, Affymetrix, Agilent, Nimblegen,
CodonDevices
Broad Inst. of Harvard MIT
PGP Volunteers Donors !
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Sequencing genomes from single cells via
polymerase clones -- Plones
(single chromosome, cell , RNA or
particle) Zhang, et al. (2006) . Nature Biotech.
June 06
1) When we only have one cell as in
Preimplantation Genetic Diagnosis (PGD)
or environmental samples (poor lab growth) 2)
Candidate chromosome region sequencing 3)
Prioritizing or pooling (rare) species based
on an initial DNA screen. 4) Multiple
chromosomes in a cell or virus 5) RNA splicing 6)
Cell-cell interactions (predator-prey,
symbionts, commensals, parasites)
Phi-29 Polymerase Stand-displacement
amplification
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Connecting molecular- micro-scales
(Beads, Bridge-PCR or Rolonies) Polymerase
-or- Ligase
147K device including computer
Shendure, Porreca, et al. 2005 Science
Mitra, et al. 1999 NAR, 2003 Analyt. Biochem.
ABI, CGI
Solexa, IBS
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Integrated Polony Sequencing Pipeline(open
source hardware, software, wetware)
In vitro paired tag libraries
Enrich amplified beads
Bead polonies via emulsion PCR
SBE or SBL sequencing
Epifluorescence Flow Cell 150K
Shendure, Porreca, Reppas, Lin, McCutcheon,
Rosenbaum, Wang, Zhang, Mitra, Church (2005)
Science 3091728.
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Matching scan time with fluidics
100M beads per sq cm
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D05 Polony Sequencing Instruments

• Automated Nikon Microscope
• Hamatsu EMCCD 9100-02
• Autosampler fluidics
• Total cost 150K including 2 computers

Rich Terry
Greg Porreca
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3
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Selective genome sequencing
7 ways to capture alleles from genomic or c-DNA
1.
2.
3.

For rearrangments
Shendure, et al. Science 309(5741)1728-32.
Nilsson et al. (2006) Trends Biotechnol 2483.
How do we optimize gt100K 100mers ?
Zhang, Chou, Shendure, Li, Leproust, Dahl, Davis,
Nilsson, Church,
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10 Mbp of oligos / 300 chip
1000X lower oligo costs

• 8K Atactic/Xeotron/Invitrogen
• Photo-Generated Acid
• 12K Combimatrix Electrolytic
• 44K Agilent Ink-jet standard reagents
• 380K Nimblegen/GA Photolabile 5'protection

Amplify pools of 50mers using flanking universal
PCR primers 3 paths to 10X error correction
Tian et al. Nature. 4321050 Carr Jacobson
2004 NAR Smith Modrich 1997 PNAS
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New in vivo genetic codes no functional DNA
exchange, phage-resistance novel amino acids
Freeing 4 tRNAs, 7 codons UAG, UUR, AGY,
AGR e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) high
serum stability
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Isaacs Church Forster Carr Jacobson
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2
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Genome engineering CAD
Recombination in human cells
Recombination in vivo E.coli
Polymerase in vitro
70b 15Kb
5Mb 250 Mb
Error Correction MutS 1E-4
Human(Artificial) Chromosomes HACs
Bacterial (Artificial) Chromosomes BACs
Chemical Synthesis 1E-2
Sequencing 1E-7
Isaacs, Carr, Emig, Gong, Tian, Reppas, Jacobson,
Church
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rE.coli Strategy 3 ss-Oligonucleotide Repair
DNA Replication Fork
Ellis et al. PNAS 2001 Constantino Court. PNAS
2003
Obtain 25 recombination efficiency in E. coli
strains lacking mismatch repair genes (mutH,
mutL, mutS, uvrD, dam)
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Multiplex Automated Genome Engineering (MAGE)
Wash with water DNA pool (50)
Concentrate
O-ring
membrane
Resuspend, bubble, select
Concentrate, electroporate
Wang, Isaacs, Terry
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Multiplex Automated Genome Engineering (MAGE)
syringe pump
computer communication /
data acquisition system
electrically actuated valves
OD sensor
electroporation cuvette w/ membrane filter
Wang, Isaacs, Terry
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ss-Oligo-Repair Experiments
Recombination Efficiency vs. Oligo Length
Recombination Efficiency vs. Oligo
observed
expected

• 90mer oligos are optimal
• Two oligos synergistic

• High recombination frequencies from 0.25 to
  25 mM oligo

Wang, Isaacs, Carr, Jacobson, Church
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Recombination-Cycling UAG-gtUAA E.coli Essential
Genes
54 oligos, 20 cycles
Mutation Distribution 11 oligos, 15 cycles
(11 loci assayed)

• Estimate complete 40 UAG ? UAA essential genes
  in 3 weeks (in assay)
• Scaling Automation
• Increase Efficiency of Recombination

Wang, Isaacs, Carr, Jacobson, Church
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Computing via combinatorial synthesis evolution
- sequencing
Second Passage
First Passage
?trp/?tyrA pair of genomes shows best co-growth
Reppas, Lin et al. Accurate Multiplex Polony
Sequencing of an Evolved Bacterial
Genome 2005 Science
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Sequence monitoring of evolution(optimize small
molecule synthesis/transport)
Sequence trp-
Reppas, Lin Church
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Co-evolution of mutual biosensors/biosynthesisseq
uenced across time within each time-point

• Independent lines of
• TrpD TyrD co-culture
• 5 OmpF (pore large,hydrophilic gt small)
• 42R-gt G,L,C, 113 D-gtV, 117 E-gtA
• 2 Promoter (cis-regulator)
• -12A-gtC, -35 C-gtA
• 5 Lrp (trans-regulator)
• 1bD, 9bD, 8bD, IS2 insert, R-gtL in DBD.
• Heterogeneity within each time-point .

Reppas, Shendure, Porecca
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(No Transcript)
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.
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Combimatrix
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Sequencing Genomes 77 to 07
'75 typed in all NA sequences '77 1st
auto-seq-reader '77 1st plasmid genome '84
Genomic Sequencing '94 1st Commercial H.pylori
'03 Polony sequencing '07 Targeted
77 ? 80G '03 93/2 3G '04
70 2M '07 93 200K '07 1
3K
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Personal Genome Project (PGP) 1K/1

• NIH CEGS submitted May 2003 approved Mar 2004
  (all but PGP)
• HMS IRB Human Subjects protocol approved Aug
  2005.
• (Possibly unique in including identifiable
  features)
• Highly-informed individuals consenting to
  potentially non-anonymous genomes extensive
  phenotypes (medical records, imaging, omics).
  Volunteer waiting list. http//pgen.us
• Cell lines in Coriell NIGMS Repository
• (B-cells, keratinocytes, fibroblasts)
• G M Church GM (2005) The Personal Genome Project
• Nature Molecular Systems Biology
  doi10.1038/msb4100040
• Kohane IS, Altman RB. (2005) Health-information
  altruists--a potentially critical resource. N
  Engl J Med. 3532074-7.
• McGuire AL, Gibbs RA (2006). Genetics. No longer
  de-identified. Science. 312370-1.

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Single-cell sequencing 4.7 Mbp (plones)

• Ultra-clean conditions for reduction of
  background amplification Real-Time monitoring

• Post-amplification chip hybridization
  distinguishes alleles
• Amplification variation random easily filled
  by PCR

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Monitoring resistance to BCR-ABL-kinase
inhibitors with polonies during CML patient
therapy Nardi, Raz, Chao, Wu, Stone, Cortes,
Deininger, Church, Zhu, Daley (submitted)
M244V
T315I
E255K
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Intelligent Design Genome Evolution

• Fong SS, Burgard AP, Herring CD, Knight EM,
  Blattner FR, Maranas CD, Palsson BO. In silico
  design and adaptive evolution of Escherichia coli
  for production of lactic acid. Biotechnol Bioeng.
  2005 91(5)643-8.
• Rozen DE, Schneider D, Lenski RE Long-term
  experimental evolution in Escherichia coli. XIII.
  Phylogenetic history of a balanced polymorphism.
  J Mol Evol. 2005 61(2)171-80
• Andries K, et al. (JJ) A diarylquinoline drug
  active
• on the ATP synthase of Mycobacterium
  tuberculosis.
• Science. 2005 307223-7.
• Shendure et al. Accurate Multiplex Polony
  Sequencing of an Evolved Bacterial Genome
  Science 2005 3091728 (Select for secretion
  altruism).