Harvard Med, WashU, MIT

NIH-CEGS, DARPA, PhRMA, DOE-GTL

Sequencing: Helicos, Ambergen, Caliper, BC-Agencourt-GTC,

Synthesis: Nimblegen, Atactic/Invitrogen, CodonDevices

Systems: BGMedicine, Genomatica

MPM Santa Barbara Thu 21-Jul-2005 1:30 PM

Synthesizing & Sequencing Genomes

1974 Code

1984 Genome Project

1994 H.pylori

2004 Synthetic Biology

3 Exponential technologies(synergistic)

Shendure J, Mitra R, Varma C, Church GM, 2004 Nature Reviews of Genetics. Carlson 2003 ; Kurzweil 2002; Moore 1965

1E-3

1E-1

1E+1

1E+3

1E+5

1E+7

1E+9

1E+11

1E+13

1830 1850 1870 1890 1910 1930 1950 1970 1990 2010

urea

E.coli

B12

tRNA

operons

telegraph

Computation &Communication

(bits/sec~m$)

Synthesis (amu/project~M$)

Analysis(kamu~base/$) tRNA

Safer biology via synthetic biology• Cell & Eco Systems modeling• HiFi automated gene replacement•Inexpensive bio-weather-map custom biosensors (airborne & medical fluids), • International bio-supply-chain licensing (min research impact, max surveillance)• Metabolic dependencies prevent survival outside of controlled environments• Multi-epitope vaccines & biosynthetic drugs.• Cells resistant to most existing viruses • via codon changes see: arep.med.harvard.edu/SBP

difficulty

Constructing new genetic codes(two examples)

1. Codons: 313 UAG stop > UAA stop2. Delete RF1 (1 free codon, for new aa e.g. PEG-pAcPhe-hGH) 1. Codons: AGY Ser > UCX Ser2. tRNAs: AGY Ser > AGY Leu3. Codons: UUR/CUX Leu > AGY4. tRNAs: UUR Leu > UUR Ser5. Codons: UCX Ser > UUR Ser (Leu & Ser now switched & 8 codons free)

Why Synthetic Genomes & Proteomes?

• Test or engineer cis-DNA/RNA-elements• Drug biosynthesis e.g. Artemesinin (malaria)• Epitopes & vaccines (HIV gag)• Unnatural aa & post-translational modifications• De novo protein design & selection.• Humanizing imm/tox systems, E.colizing codons • 20 bit in vivo counters

•Why whole genomes?Changing the genetic code, safety, genome stability, enhanced restriction, recombination

10 Mbp of oligos / $1000 chip

<1K Oxamer Electrolytic acid/base 8K Atactic/Xeotron/Invitrogen Photo-Generated Acid Sheng , Zhou, Gulari, Gao (U.Houston) 12K Combimatrix Electrolytic 24K Agilent Ink-jet standard reagents380K Nimblegen Photolabile 5'protection Nuwaysir, Smith, Albert

Tian et al. Nature. 432:1050

~1000X lower oligo costs

(= 2 E.coli genomes or 20 Mycoplasmas /chip)

Improve DNA Synthesis CostSynthesis on chips in pools is 1000X 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 remove tags.

10 50 10 => ss-70-mer (chip)

20-mer PCR primers with restriction sites at the 50mer junctions

Tian, Gong, Sheng , Zhou, Gulari, Gao, Church

=> ds-90-mer

=> ds-50-mer

Improve DNA Synthesis Accuracyvia mismatch selection

Tian & Church Other mismatch methods: MutS (&H,L)

Improving DNA synthesis accuracy

Method Bp/error

Chip assembly only 160 Hybridization-selection 1,400MutS-gel-shift 10,000PCR 35 cycles 10,000MutHLS cleavage 100,000In vivo replication 1,000,000,000

0 MutS

2 MutS

Tian & Church 2004 NatureCarr & Jacobson 2004 NAR

Smith & Modrich 1997 PNAS{

CAD-PAM: Computer-Aided Design -Polymerase Assembly Multiplexing

For tandem, inverted and dispersed repeats: Focus on 3' ends, hierarchical assembly, size-selection and scaffolding.

Mullis 1986 CSHSQB, Dillon&Rosen 1990 BioTechniques, Stemmer 1995 Gene, Smith et 2003 PNAS, Tian et al. 2004 Nature,

50

75

125 225 425 825 … 100*2^(n-1)

E.coli genome synthesis pipeline

0 1 2 3 4 PAM cycle# 550 75 125 225 425 #bp 825

50 HS PAM 425 MutS PAM 10K anneal 100K red5Mbp

USER-T4Pol USER-T4Pol USER-5'only 1 pool 480 pools 480 genomic 48 1 of 117K universal primers primer pairs

HS=Hybridization-SelectionUSER=Uracil DNA glycosylase &EndoVIII to remove flanking primer pairs

PCR in vitro

Isaacs, Carr, Emig, Gong, Tian, Reppas,

Jacobson, Church

in vivo

>3 days >1 day ~1day >7days

5 Mbp Genome assembly alternatives

1. cat

Automated in vivo homologous recombination:Serial electroporation: 48 stages: 1 strain (21 hr/stage)vs. Hierarchical conjugation: 7 stages: 48 > 24 > 12 > 6 > 3 > 2 > 1 strainsvs. Random/simultaneous 1 or more stages

3. cat2. kan

Reppas & Church

All 30S-Ribosomal-protein DNAs(codon re-optimized)

Tian, Gong, Sheng , Zhou, Gulari, Gao, Church

1.7 kb

0.3 kb

s190.3kb

Nimblegen 95K chip

Atactic <4K chip 14.6 kb

Full codon remapping for enhanced protein expression

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, lower %GC

Tian et al. Nature. 432:1050

20w 20m 17w 17m 16w 16m

10kd

W: wild-typeM: modified

Western blot based on His-tags

Genome engineering: safety via metabolic dependency

trp/tyrA pair of genomes shows the best co-growth

Reppas, Lin & Church unpublished.

First Passage SecondPassage

(ideally ecologically uncommon molecules)

0

1

2

3

4

5

6

7

8

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

# of passages

Do

ub

lin

g t

ime

(h

r)

Q1

Q3

Q2-1

Q2-2

EcNR1

Sequence monitoring of evolution(anticipate escape & resistance)

Molecular Genomic Imaging Center (CEGS)Harvard / Wash U

George Church, Rob MitraGreg Porreca, Jay Shendure

Sequencing by Ligation on Polony Beads

with Nick Reppas, Kun Zhang, Shawn Douglas, Mike Wang,

Abraham Rosenbaum, Agencourt

Personal Genomics, Stem Cells, ELSI

Synthetic Biology

A’

A’A’

A’

A’

A’

B

BB

B

BB

A

Single molecule from a library or population

B

BA’

A’

Primer is Extendedby Polymerase

B

A’

BA’

Polymerase colony (polony) PCR in a gel (or emulsion-beads)

Primer A has 5’ immobilizing Acrydite (or double biotin to SA-beads)

Mitra & Church Nucleic Acids Res. 27: e34

New Polony Sequencing Pipeline

In vitro paired tag libraries

Bead polonies via emulsion PCR

Monolayer gel

immobilization

Enrich amplified

beads

SOFTWARE

Images → Tag Sequences

Tag Sequences → GenomeSBE or SBLsequencing

Epifluorescence & Flow Cell

Mitra, Shendure, Porreca, Rosenbaum, Church unpub.

~1 kb genomic fragment

paired genomic tags

(17 to 18 bp each)

common sequences

MmeI

Fisseq-F Fisseq-RT30 Tag 2Tag 1Fisseq-FLeft RightMid Seq2Seq1

In vitro construction of a complex, mate-paired library

43 bp 32 25

Total = 134-136 bp amplicon

Enrichment by Hybridization

Selector Bead

One of 750 megapixel frames of gel-immobilized 1.0 micron beads, 0.3 micron pixels, 4-colors

ACUCAUC…(3’)…TAGAGT????????????????TGAGTAG…(5’)

5’-Cy5-nnnnAnnnn-3’ 5’-Cy3-nnnnGnnnn-3’ 5’-TR-nnnnCnnnn-3’ 5’-Cy3+Cy5-nnnnTnnnn-3’

5'PO4

Sequencing by Ligation (SBL) with fluorescent combinatorial 9-mers

Excitation Emission 647 700 555 605 572 630 555 700

nm

Consensus Accuracy False Positives (E.coli) False Positives (Human)1E-3 4,000 3,000,000

1E-4 BERMUDA/ABI 400 300,000

1E-6 Polony-SBL 4 3000

Goal of Resequencing Discovery of Uncommon VariationE.g. cancer mutations <1E-6

Why low error rates?

ABI 2004 Jun 2005 2006 >2007

# bp/expt - 2e7 3e7 3e8 60e9

Complexity (bp) - 74 4e6 3e9 6e9

Avg Fold Cov 8 3e5 6 0.1 10

Pix per bp - 300 1724 333 1Read-length 900 14 (SBE) 25 (pair) 35 42

$ / kb (e<1e-5) 2.4 - .24 .12 2e-5

$/ 1X 3e9 b 2e6 - 2e5 5e4 200

Indel Error 5e-3 0.6% 1e-3 1e-3 1e-3

Subst Error 4e-3 4e-6 1e-3 1e-3 1e-3

3X Cons Err 1e-4 - 1e-6 3e-7 1e-7

Kb / min 0.8 360 27 1e3 1e6

Pix / sec - 2e5 2e6 6e6 2e7Enz $/mg - 8 8 8 0.4

Sequencing cost (10 to 100,000 fold improvements)

Position Type Gene LocationABI

Confirmation

Comments

986,334 T > G ompF TATA box Only in evolved strain

931,960 8 bp del lrp frameshift Only in evolved strain

1,976,500 776 bp del insB_5 IS element MG1655 heterogeneity

3,957,960 C > T ppiC 5' UTR MG1655 heterogeneity

4,654,533 T > C cI Glu > Glu heterogeneity

4,647,960 T > C ORF61 Lys > Gly heterogeneity

985,797 T > G ompF Glu > Ala (in progress)

454,864 T > C tig Gly > Gly(in progress)

4,648,691 G > A exo Phe > Phe(in progress)

Mutation Discovery in Engineered/Evolved E.coli

rs3778973

rs1557917

rs39284

rs10500042

rs4717028

GM10835

C

G

C

G

C

T

A

T

A

T

TT=137 CT=2 (TC=1) CC=131

153Mb

Single human chromosome molecules(polony sequencing)

Genome Analysis Policy

• Insurance/employment: What probability & level of advantage can be hidden/examined?

• Individual/group stigma

• Choice, stem cells, cloning

• Privacy & transparency

NHGRI/DOE ELSI, Genetic Screening Study Group

Anonymity, privacy, disclosure, identity

"Open-source" meets Personal Genome-Phenome Project

• Are information-rich resources (e.g. facial imaging & genome sequence) really anonymous?

• What are the risks and benefits of "open-source"?

• What level of training is needed to give informed consent on open-ended studies?

• Harvard Medical School IRB Human Subjects protocol submitted 16-Sep-2004.

.