# Presentation: 177913

## Analysis & Synthesis of Omes  

*Thanks to:*
- _Agencourt_, Ambergen, _Atactic_, BeyondGenomics, Caliper,  Genomatica,  Genovoxx, Helicos, MJR, NEN, _Nimblegen_, ThermoFinnigan,  Xeotron/Invitrogen
**For more info see:  arep.med.harvard.edu**

-    DOE
- Wed 3-Nov-2004  11:30 AM

**Analysis ****&**** Synthesis of Omes **

## Systems Biology Loop

**Syntheses &**
**Perturbations**

**Models**

**Experimental **
**designs**

**(Systematic)**
**Data**

- Proteasome targeting
- Genome engineering

**Metabolic optimality**

**Flux & **
**Competitive growth**

**DNA**** ****&**** ****RNA**
**Polony-Seq**

**Synthetic Biology Tools**

## DOE  Synthetic Genomes: Why?

- Cheaper/faster "standard biology",  hypothesis testing
- Systems Biology: Multiple simultaneous tests
- Viruses: Aid strain transfer; generate variants, new haplotypes
- Anti-viral vaccines and therapeutics (including variants)
- In vitro: Make products toxic in E.coli.
- Microbes: Interspecific hybrids (e.g. codon usage)
- Structural biology: variants
- Rapid vaccine response to engineered bioterrorism.
- Cell-mediated immunity + humoral.
- Fix mismatch between genome analysis & synthesis

## DOE  Synthetic Genomes: Why?

- In vitro
- Microbial & Human Antimutators
- Artificial ecosystems (laboratory scales)
- Energy aiding pathway improvement
- Instrustrial production: Enzymes, SingleCellProtein, Protein-drugs
- Remediation: Hybrid genomes (opt. codons), combinatorial pathway  (Maxygen & Diversa).  Xylose & Oil
- Pharmaceuticals: Combinatorial syntheses
- Nano science  Combinatorial syntheses, Complex nanosystems, more general nanoassembly (in reach of polymerases and ribosome-like factories)
- Health research: **10X faster results per current $**  (cost/benefit)
- Hypothesize & test unknown gene combinations
- Synthetic standards (arrays, MS, quantitation, etc)
- Agriculture: salt, cold, drought, pest tolerant hybrid genomes

## Motif Co-occurrence, comparative genomics, RNA clusters, and/or  ChIP2-location data

**P= 10****-6 ****to 10****-11**

**Genome Res. 14:201–208**
**Bulyk, McGuire,Masuda,Church**

## Synthetic testing of DNA motif combinations

- 1.3                             **2.4** (1.3 in argR)
-        1.1                       1.3
-                     0.7                            **2.5**
-           **0.2**                           1.4
-             1.4                        **3.5**

**RNA Ratio (motif- to wild type) for each flanking gene **

**Bulyk, McGuire,Masuda,Church  ****Genome Res. 14:201–208**

## Synthetic Genomes & Proteomes. Why?

-  Test or engineer cis-DNA/RNA-elements
- Access to any protein (complex) including
-      post-transcriptional modifications
-  Affinity agents for the above.
-  Mass spectrometry standards, protein design
-  Utility of molecular biology DNA-RNA-Protein
-     *in vitro* "kits" (e.g. PCR, SP6, Roche)
- Toward these goals design a chassis:
-  115 kbp genome. 150 genes.
-  Nearly all 3D structures known.
-  Comprehensive functional data.

## (PURE) translation utility 

**Removing tRNA-synthetases, **
**translational release-factors,**
**RNases & proteases **
- Selection of _scFvs_ specific for HBV DNA polymerase using **ribosome display.** Lee et al. 2004 J Immunol Methods.  284:147
- Programming **peptidomimetic syntheses** by translating genetic codes designed de novo. Forster et al. 2003 PNAS 100:6353
- High level cell-free expression & specific labeling of **integral membrane proteins. **Klammt et al. 2004 Eur J Biochem  271:568
- Cell-free translation reconstituted with purified components.     Shimizu et al. 2001 Nat Biotechnol.  19:751-5.

##  in vitro genetic codes

- 5'

- Second base

- 3'

**U**

**A**

**C**

**C**

**U**

- mS

- yU

- eU

**A**

**C**

**U**

**G**

**A**

- Forster, et al. (2003) PNAS 100:6353-7

**80%** average yield
- per unnatural coupling.

- bK =  *biotinyl*lysine ,
- mS = O*methyl*serine
- eU=2-amino-4-pent*en*oic acid
- yU = 2-amino-4-pent*yn*oic acid

##     Mirror world  :  enzyme, parasite, & predator resistance& access 2n diastereomers (n chiral atoms)

- L-amino acids & D-ribose (rNTPs, dNTPs)
- Transition: EF-Tu, peptidyl transferase, DNA-ligase
- D-amino acids & L-ribose (rNTPs, dNTPs)

- Dedkova, et al.  (2003) Enhanced D-amino acid incorporation into protein by
- modified ribosomes. *J Am Chem Soc ***125**, 6616-7

## Oligos for 150 & 776 synthetic genes(for E.coli minigenome &  M.mobile whole genome respectively)

**Forster & Church**

**Oligos for ****150 & 776 synthetic genes****(for *****E.coli***** minigenome &  *****M.mobile***** whole genome respectively)**

## Up to 760K Oligos/Chip18 Mbp  for $700 raw (6-18K genes)

**Up to 760K Oligos/Chip****18 Mbp  for $700 raw (6-18K genes)**

**  ****<1K  Oxamer  **Electrolytic acid/base
**    ****8K  **_**Atactic/Xeotron/Invitrogen**_**  **Photo-Generated Acid
-           Sheng , Zhou, Gulari, Gao (U.Houston)
**  ****24K  Agilent   **Ink-jet standard reagents
**  ****48K  Febit  **
**100K  Metrigen **
**380K  **_**Nimblegen**_** **Photolabile 5'protection
-            Nuwaysir, Smith, Albert

- Tian, Gong, Church

## Improve DNA Synthesis Cost

- Synthesis on chips in pools is _**5000X**_** **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 release them.

- 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 Accuracyvia mismatch selection

**Improve DNA Synthesis **_**Accuracy**_**via mismatch selection**

- Tian & Church

- Other mismatch methods: MutS (&H,L)

## Genome assembly

- Moving forward:
- 1. Tandem, inverted and dispersed repeats
-    (hierarchical assembly, size-selection and/or scaffolding)
- 2. Reduce mutations (goal <1e-6 errors) to reduce # of intermediates
- 3. >30 kbp  homologous (Nick Reppas)
- 4. Phage integrase site-specific recombination, also for counters.
- Stemmer et al. 1995. Gene 164:49-53;Mullis 1986 CSHSQB.

- 50
- 75
- 125     225   425   825    ...  100*2^(n-1)

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

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

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

- 1.7 kb

- 0.3 kb

## Improving synthesis accuracy 9-fold

| Method | Total bp | # Clones | Trans-ition | Trans-version | Deletion | Addition | Bp/error |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Hyb  selection, PCR | 23641 | 9 | 7 | 3 | 5 | 2 | 1391 |
| Gel selection, PCR | 24546 | 35 | 28 | 12 | 11 | 3 | 455 |
| No selection, ligation
+PCR | 6093 | 25 | 6 | 6 | 22 | 4 | 160 |
| No selection, PCR | 9243 | 21 | 25 | 13 | 19 | 1 | 159 |

- Tian & Church

## Extreme mRNA makeover for protein expression in vitro

**Extreme mRNA makeover**** ****for protein expression *****in vitro***

- 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.**

- Tian & Church

- Western blot based on His-tags

## Systems Biology Loop

**Syntheses &**
**Perturbations**

**Models**

**Experimental **
**designs**

**(Systematic)**
**Data**

- Proteasome targeting
- Genome engineering

**Metabolic optimality**

**Flux & **
**Competitive growth**

**DNA**** ****&**** ****RNA**
**Polony-Seq**

**Synthetic Biology Tools**

## Why sequence?

- • **Cancer:** mutation sets for individual clones, loss-of-heterozygosity
- • **Pathogen "weather map",** biowarfare sensors
- • **RNA splicing** & chromatin modification patterns.
** ****Synthetic biology **& lab selections
-  Antibodies or "aptamers" for any protein
-  B & T-cell receptor diversity: Temporal profiling, clinical
-  Preventative medicine & genotype–phenotype associations
-  Cell-lineage during development
-  Phylogenetic footprinting, biodiversity

**Shendure et al. 2004  ****Nature Rev Gen ****5, 335.**

## Sequencing single molecules

- Ecosystem studies really need single-cell amplification because of  multiple chromosomes  (& RNAs)

- (Even an 80% genome coverage is  better than 100 kb BACs)

## Single bacterial chromosome amplification

- Ratio to unamplified hybridization
- along the
- chromosome of
*Escherichia* & *Prochlorococus*
- on
- Affymetrix chips.

## Convergence on non-electrophorectic tag sequencing methods?

**Tag**     >400   14-26	  20	   100    26 bp (2-ends)
-             EST   SAGE    MPSS   454   Polony-Seq
-  Single-molecule vs. amplified single molecule.
-  Array vs. bead packing vs. random
-  Rapid scans vs. long scans (chemically limited, 454)
-  Number of immobilized primers:
-    0: Chetverin'97 "Molecular Colonies"
-    1: Mitra'99 > Agencourt  **"Bead Polonies"**
-    2: Kawashima'88, Adams'97 > Lynx/Solexa: "Clusters"

- http://arep.med.harvard.edu/Polonator/Plone.htm

## Polony Fluorescent In Situ Sequencing Libraries 

**Polony **_**F**_**luorescent **_***I***_***n ***_***S***_***itu***** **_**Seq**_**uencing ****Libraries **

- Greg Porreca
- Abraham Rosenbaum

**1 to 100kb Genomic**

**M**

**L**** ****       ****R**
**M**

- PCR
- bead

- Sequencing
- primers

**2x20bp after MmeI (****BceAI, AcuI)**

- Dressman et al PNAS 2003 emulsion

## Cleavable dNTP-Fluorophore (& terminators)

- Cleavable dNTP-Fluorophore (& terminators)

- Mitra,RD, Shendure,J, Olejnik,J, Olejnik,EK, and Church,GM (2003) Fluorescent in situ Sequencing on Polymerase Colonies. Analyt. Biochem. 320:55-65

- Reduce
- or
- photo-
- cleave

**Notes:**
Figure 3.  (A)  Structure of Cy5-SS-dCTP.  This molecule has a disulfide bond between the fluorophore and the deoxynucleotide so that the label can be removed after incorporation.  (B)  Extension of an immobilized primer:template with Cy5-dCTP and Cy5-SS-dCTP.  Both molecules are efficiently and specifically incorporated.  After a wash in 5mM DTT, the fluorescence was removed from the slide extended with Cy5-SS-dCTP, but not the slide extended with Cy5-dCTP. 

## Polony-FISSeq:  up to 2 billion beads/slide

**0.5% of full gel area**

**Polony-FISSeq:  up to 2 billion beads/slide**

## Polony-FISSeq:  up to 2 billion beads/slide

**Polony-****FISSeq****:****  up to 2 billion beads/slide**
**Cy5 primer (570nm) ; ****Cy3 dNTP (666nm)**

- Jay Shendure

## # of bases sequenced (total)	23,703,953
# bases sequenced (unique)	73
Avg fold coverage			324,711 X
Pixels used per bead (analysis)	~3.6
Read Length per primer		14-15 bp
Insertions            			0.5%          
Deletions             			0.7%          
Substitutions   (raw)   		4e-5   
Throughput:			360,000 bp/min         

- # bases sequenced (unique)**	****73**
- Avg fold coverage	**	****	****324,711 X**
- Pixels used per bead (analysis)	~**3.6**
- Read Length _per primer_**	****	****14-15 bp**
- Insertions            			**0.5%**
- Deletions             			**0.7%**
- Substitutions**  ** (raw)   		_**4e-5**__ _
- Throughput:	**	****	**_**360,000**_** bp/min**

**Polony FISSeq  Stats**

- Current capillary sequencing  _**1400**_** bp/min**
- (_**600X**_ speed/cost ratio,  ~$5K/1X)
**(This may omit: PCR , homopolymer, context errors)**

- Shendure

**Notes:**
$1/600b ABI /600 = $8K/3e9
 $1e5/(5yr*365*24*60) / 4e5 bp/min = $300/1X

## Systems Biology Loop

**Syntheses &**
**Perturbations**

**Models**

**Experimental **
**designs**

**(Systematic)**
**Data**

- Proteasome targeting
- Genome engineering

**Metabolic optimality**

**Flux & **
**Competitive growth**

**DNA**** ****&**** ****RNA**
**Polony-Seq**

**Synthetic Biology Tools**

## .

## High accuracy special case: homopolymers (e.g. AAA, CC, etc.)

- Use "compressed" tags ,  ACG = ACCG=ACCCG
- Quantitate incorporation
- Reversible terminators
- "Wobble sequencing"
-      All of these work.
- Maintenance of amplification fidelity using linear amplification from initial genomic fragment

## "Wobble sequencing" for homopolymers

- 6 positions * 16 primers  *   4 dNTPs    => 13 bp (paired ends)
- CCTCATTCTCT AA   + dATP (then C, ...)
- CCTCATTCTCT AC   + dATP (then C, ...)
- . . .
- CCTCATTCTCTnnAA  + dATP (then C, ...)
- . . .
- CCTCATTCTCTnnNNnnNNnnTT  + dATP (then C, ...)
- 4.5/64  bp/cycle (for wobble sequencing)   vs.
-  2.5/4   bp/cycle (for simple sequential base-extension)