Dinh 2011/NOTES/2011-11-4

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Multiplexed Bisulfite Patch PCR[edit]

File:Bisulfite Patch PCR - Design.pdf

  • Needs/Objective:

Given a set of targets of various sizes, find the best compatible enzymes and patch oligos to achieve maximal coverage of the targets.

Goals
1) Find best restriction digest of genomic DNA: 
   Minimum number of enzymatic steps to reach every target at a sequenceable distance
2) Maximal coverage of the targets
   >95% coverage
3) 10,000 samples in reasonable time
   Multiplex
   Reduce the number of steps before pooling samples together.
4) Low input 
  • Program
1) Input target set
2) Obtain target sequences +/- 50bp
3) Build compatible RE's and respective recognition sequence database
4) Search through target sequences for RE recognition sites:
   Mark RE recognition sites with known SNPs
5) Exhaustively iterate through all possible compatible RE recognition sequence(s) and score each output:
   Let Re = {set of compatible enzymes}
   Let Z = 0 if a RE recognition sequence has a SNP; 1 otherwise
   Score function,
   S (Re) = Z * total sequenceable target (%) / total number of enzymes needed
6) Return Re with maximal score
7) Return patch oligos:
   "Patch" sequence must be 20-22 bp long, and such that all patch sequences must have melting temperatures greater than 60C
  • In progress script
File:MyTemplateDigest.txt
File:NEB Buffer Chart.txt
File:NEB Cutsite Chart.txt
  • Tested on gene body of HRAS with 50bp ext.
  • Outputs:
recognition_site #numberOfSnps
cut_pos: enzymes
ie.

CCCGG	4
0|BssKI;StyD4I
2|ScrFI;NciI
AATT	0
0|MluCI
CCATGG	0
0|NcoI;StyI;BtgI;BsaJI
CTAG	0
1|BfaI
GAGCTC	0
2|Ecl136II;Eco53KI
4|SacI;BanII;BsiHKAI;Bsp1286I
AGCGCT	1
2|AfeI
4|HaeII
.
.
.