Dinh 2011/NOTES/2011-11-4: Difference between revisions
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==Multiplexed Bisulfite Patch PCR== | ==Multiplexed Bisulfite Patch PCR== | ||
[[File: | [[File:Bisulfite Patch PCR - Design.pdf]] | ||
*'''Needs/Objective:''' | *'''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. | Given a set of targets of various sizes, find the best compatible enzymes and patch oligos to achieve maximal coverage of the targets. |
Revision as of 20:28, 4 November 2011
Multiplexed Bisulfite Patch PCR
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