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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 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’).