Transcript Document 7267336
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 PGP Volunteers & Donors !
Broad Inst. of Harvard & MIT
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
Connecting molecular- & micro-scales
$147K device including computer (Beads, Bridge-PCR or Rolonies)
Polymerase
-or-
Ligase
A
Shendure, Porreca,
et al. 2005 Science
Mitra, et al.
1999 NAR, 2003 Analyt. Biochem.
G C T
ABI, CGI Solexa, IBS
Integrated Polony Sequencing Pipeline
(open source hardware, software, wetware)
In vitro paired tag libraries Bead polonies via emulsion PCR Enrich amplified beads Monolayer gel immobilization SOFTWARE → 100G Images → 2G Tag Sequences 300K Genome diffs SBE or SBL sequencing Epifluorescence &
Flow Cell $150K
Shendure, Porreca,
Reppas, Lin, McCutcheon, Rosenbaum, Wang, Zhang, Mitra, Church (2005) Science 309:1728.
Matching scan time with fluidics
100M beads per sq cm
D05 Polony Sequencing Instruments
•Automated Nikon Microscope •Hamatsu EMCCD 9100-02 •Autosampler & fluidics Total cost: $150K including 2 computers
1
Greg Porreca
2 3
Rich Terry
4 5 6
Selective genome sequencing
7 ways to capture alleles from genomic or c-DNA 1.
In vitro Paired-tag library
2.
Gap fill
3.
Cleave & ligate
For rearrangments
Red=Synthetic; Yellow=genome/cDNA
Shendure, et al. Science 309(5741):1728-32. Nilsson et al. (2006) Trends Biotechnol 24:83.
How do we optimize >100K 100mers ?
4. Hybridize-select 5. Allelic-RNA-ratio 6. Mb region primers 7. Dilution haplotype
Zhang, Chou, Shendure, Li, Leproust, Dahl, Davis, Nilsson, Church,
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. 432:1050 Carr & Jacobson 2004 NAR Smith & Modrich 1997 PNAS
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
TTT TTC TTA TTG CTT CTC CTA CTG ATT ATC ATA ATG GTT GTC GTA GTG L I M V F 30362 22516 L
4
18932 18602 15002 15077 5314 71553 41309 34178 5967 37915 24858 20753 14822 35918 TCT TCC TCA TCG CCT CCC CCA CCG ACT ACC ACA ACG GCT GCC GCA GCG S P T A 11495 11720 9783 12166 9559 7485 11471 31515 12198 31796 9670 19624 20762 34695 27418 45741 TAT TAC TAA TAG CAT CAC CAA CAG AAT AAC AAA AAG GAT GAC GAA GAG Y H 21999 16601 STOP STOP
1
2703 326 17613 Q N K D E 13227 20888 39188 24159 29385 45687 14029 43719 25918 53641 24254 TGT TGC TGA TGG CGT CGC CGA CGG AGT AGC AGA AGG GGT GGC GGA GGG C STOP W R S R G 11970
3
21862
2
2896 1692 33622 40285 10893 15090 7048 8816 1256 20683 28382 29898 4859 7399
Isaacs Church Forster Carr Jacobson
Genome engineering CAD
Polymerase
in vitro
Recombination
in vivo E.coli
70b 15Kb
Recombination in human cells
5Mb 250 Mb Chemical Synthesis
1E-2
Error Correction MutS
1E-4
Bacterial (Artificial) Chromosomes Sequencing
1E-7
BACs Human(Artificial) Chromosomes HACs Isaacs, Carr, Emig, Gong, Tian, Reppas, Jacobson, Church
rE.coli Strategy #3: ss-Oligonucleotide Repair DNA Replication Fork
Obtain 25% recombination efficiency in
E. coli
strains lacking mismatch repair genes (
mutH, mutL, mutS, uvrD, dam)
Ellis et al. PNAS 2001 Constantino & Court. PNAS 2003
Improved Recombination Frequency: 10 -4
0.25 (> 3 log increase!)
Multiplex Automated Genome Engineering (MAGE)
Wash with water & DNA pool (50) Concentrate, electroporate Concentrate O-ring membrane Resuspend, bubble, select Wang, Isaacs, Terry
Multiplex Automated Genome Engineering (MAGE)
syringe pump computer communication / data acquisition system electrically actuated valves electroporation cuvette w/ membrane filter Wang, Isaacs, Terry OD sensor
ss-Oligo-Repair Experiments Recombination Efficiency vs. Oligo Length observed expected Recombination Efficiency vs. [Oligo]
90mer oligos are optimal Two oligos synergistic
High recombination frequencies from 0.25 to 25
m
M oligo
Wang, Isaacs, Carr, Jacobson, Church
Recombination-Cycling UAG -> UAA E.coli Essential Genes Mutation Distribution: 11 oligos, 15 cycles
25 20 15 10 5 0 0 1 2 3 4 5
# mutations/clone
6 7
54 oligos, 20 cycles
40 30 20 10 0
(11 loci assayed)
0 1 2 3
# mutations/clone
4 5
Oligo Pool 11 54 # cycles Best Clone (98 %tile) 15 20 7 16 (estimate) Fraction of mutated sites 7/11 16/54 Time 5 days 7 days
Estimate: complete ~40 UAG
UAA essential genes in ~3 weeks (in assay) Scaling & Automation Increase Efficiency of Recombination
Wang, Isaacs, Carr, Jacobson, Church
Computing via combinatorial synthesis – evolution - sequencing
First Passage Second Passage
trp/
tyrA growth pair of genomes shows best co-
Reppas, Lin et al. ; Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome 2005 Science
Sequence monitoring of evolution (optimize small molecule synthesis/transport)
8 7 6 5 4 3 2 Sequence trp Q1 Q3 Q2-1 Q2-2 EcNR1 1 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
# of passages
Reppas, Lin & Church
Co-evolution of mutual biosensors/biosynthesis sequenced across time & within each time-point Independent lines of Trp
& Tyr
co-culture
5 OmpF: (pore: large,hydrophilic > small) 42R-> G,L,C, 113 D->V,
117 E->A
2 Promoter: (cis-regulator) -12A->C, -35 C->A 5 Lrp: (trans-regulator) 1b , 9b , 8b , IS2 insert, R->L in DBD.
Heterogeneity within each time-point .
Reppas, Shendure, Porecca -12 -11 -10 -9 -8 -7 -6
.
Combimatrix
Sequencing Genomes ’77 to ‘07
'75 typed in all NA sequences '77 1 st auto-seq-reader '77 1 st plasmid genome '84 ‘Genomic Sequencing’ '94 1 st Commercial
H.pylori
'03 Polony sequencing '07 Targeted
’77 ? % $80G '03: 93 % /2 $3G '04 70% $2M '07: 93 % $200K '07: 1 % … $3K
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
doi:10.1038/msb4100040 Kohane IS, Altman RB. (2005)
Health-information altruists--a potentially critical
resource. N Engl J Med. 353:2074-7. McGuire AL, Gibbs RA (2006).
Genetics. No longer de-identified
. Science. 312: 370-1.
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
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
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
balanced polymorphism. Long-term experimental evolution in Escherichia coli. XIII. Phylogenetic history of a
J Mol Evol. 2005 61(2):171-80 Andries K, et al. (J&J)
A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis.
Science. 2005 307:223-7.
Shendure et al.
Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome
Science 2005 309:1728
(
Select for secretion & ‘altruism’).