Document 7267336

Report
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.
PGP Volunteers & Donors !
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
Mitra, et al.
1999 NAR,
2003 Analyt.
Biochem.
G
C
T
ABI, CGI
Solexa, IBS
Shendure,
Porreca, et al.
2005 Science
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
SBE or SBL
sequencing
Epifluorescence &
Flow Cell $150K
100G Images →
2G Tag Sequences
→ 300K Genome diffs
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
Rich Terry
Greg Porreca
1
2
3
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.
4. Hybridize-select
5. Allelic-RNA-ratio
6. Mb region primers
7. Dilution haplotype
How do we optimize >100K 100mers ?
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
F
TTA
30362
TCT
11495
TAT
22516
TCC
11720
TAC
18932
TCA
9783
S
21999
TGT
Y
16601
TGC
C
8816
TAA
STOP
2703
TGA
STOP
1256
STOP
326
TGG
W
20683
17613
CGT
28382
13227
CGC
29898
20888
CGA
39188
CGG
7399
24159
AGT
11970
29385
AGC
45687
AGA
14029
AGG
43719
GGT
25918
GGC
4 18602
TCG
12166
TAG
CTT
15002
CCT
9559
CAT
CTC
15077
CCC
7485
CAC
CTA
5314
CCA
11471
CAA
71553
CCG
31515
CAG
41309
ACT
12198
AAT
34178
ACC
31796
AAC
5967
ACA
9670
AAA
37915
ACG
19624
AAG
24858
GCT
20762
GAT
20753
GCC
34695
GAC
TTG
CTG
L
L
ATT
ATC
I
ATA
ATG
M
GTT
GTC
V
P
T
H
Q
N
K
D
1
A
7048
R
4859
S
3 21862
R
2 1692
2896
33622
40285
G
GTA
14822
GCA
27418
GAA
GTG
35918
GCG
45741
GAG
E
53641
GGA
10893
24254
GGG
15090
Isaacs
Church
Forster
Carr
Jacobson
Genome engineering CAD
Recombination
in vivo E.coli
Polymerase
in vitro
70b
15Kb
Error
Correction
MutS 1E-4
Chemical
Synthesis
1E-2
5Mb
Bacterial (Artificial)
Chromosomes
BACs
Recombination
in human cells
250 Mb
Human(Artificial)
Chromosomes
HACs
Sequencing
1E-7
Isaacs, Carr, Emig, Gong, Tian, Reppas, Jacobson, Church
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)
Improved Recombination Frequency:
10-4  0.25 (> 3 log increase!)
Multiplex Automated Genome Engineering (MAGE)
Wash with
water &
DNA pool
(50)
Concentrate
O-ring
Concentrate,
electroporate
Wang, Isaacs, Terry
membrane
Resuspend,
bubble,
select
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
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
Recombination-Cycling UAG->UAA E.coli Essential Genes
54 oligos, 20 cycles
40
25
20
15
10
5
0
Frequency
Frequency
Mutation Distribution: 11 oligos, 15 cycles
(11 loci assayed)
30
20
10
0
0
1
2
3
4
5
6
7
0
# mutations/clone
1
2
3
4
5
# mutations/clone
Oligo
Pool
# cycles
Best Clone (98 %tile)
Fraction of mutated sites
Time
11
15
7
7/11
5 days
54
20
16 (estimate)
16/54
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
Second
Passage
First
Passage
trp/tyrA pair of genomes shows best cogrowth
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
Doubling time (hr)
7
6
5
Q1
Q3
4
Q2-1
Q2-2
3
Sequence trp-
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 1st auto-seq-reader
'77 1st plasmid genome
'84 ‘Genomic Sequencing’
'94 1st Commercial H.pylori
'03 Polony sequencing
'07 Targeted
’77
'03:
'04
'07:
'07:
?%
93%/2
70%
93%
1%
…
$80G
$3G
$2M
$200K
$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 nonanonymous 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 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. (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’).

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