Dinh 2011/NOTES/2011-11-4: Difference between revisions
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>Dinh m (Created page with '==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 p…') |
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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. | ||
Goals | Goals | ||
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4) Low input | 4) Low input | ||
'''Program''' | *'''Program''' | ||
1) Input target set | 1) Input target set | ||
2) Obtain target sequences +/- 50bp | 2) Obtain target sequences +/- 50bp | ||
Line 25: | Line 25: | ||
S (Re) = Z * total sequenceable target (%) / total number of enzymes needed | S (Re) = Z * total sequenceable target (%) / total number of enzymes needed | ||
6) Return Re with maximal score | 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 | |||
. | |||
. | |||
. |
Latest revision as of 16:23, 7 November 2011
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 . . .