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 . . .