Kun:LabNotes/CpgSeq
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2008 <calendar> name=Kun:LabNotes/CpgSeq format=%name/%year-%month-%day date=2008/02/01 view=oneyear </calendar> 2007 <calendar> name=Kun:LabNotes/CpgSeq format=%name/%year-%month-%day date=2007/11/01 view=threemonths </calendar>
Probe design
Agilent probes, V2 (April 2008)
- Why redesigning probes?
- V1 design was based on Primer3. Many constrains in Primer3 are probably either unnecessary or inappropriate for padlock probes.
- We would like to search for an optimal (maximal coverage, minimal number of CpG in H1 & H2) set of probes covering a CpG island. It is hard to integrate Primer3 into the search of such an optimal set.
- The capturing sequences should be as specific as possible. They don't have to be completely unique however, since a low percentage of non-specific capture is acceptable.
- What are the differences in the V2 algorithm?Code.
- The codes for the search of capturing sequences were completely rewritten.
- The algorithm first search for all oligos with appropriate length and Tm, then look for pairs with appropriate gap size among all pair-wise combinations of these oligos.
- These pairs were assigned scores based on criteria including Tm, size, gap size, number of CpGs and a frequency score based on word statistics (word size K=12).
- Because the number of such pairs could be very large, the target region was then divided into a number of 10-bp bins, and only the pair with the best score in each bin was selected for the next round of optimization.
- These pairs are then assembled into sets. These sets cover every combination of the pairs. Each set has a score, which is based on the score of each pairs, plus the coverage of all pairs in the set.
- The candidate sets are then sorted based on the score, and the one with the best score was selected.
- To evaluate the specificity of capturing sequences, the counts of all 12-mers in both strands of the bisulfite converted genome were calculated, and saved as a data file. This file was loaded into the memory during probe design. The frequency score of each sequence is a weighted sum of all 12-mer sub-sequences. This calculation has a directionality, because the specificity for the sub-sequence close to the gap (for ligation or extension) should be higher. So the weight for the first 12-mer next to the gap is 1, and it is reduced by 25% per base as the sub-sequence moves outwards. I also added a hash table as a buffer so that the same oligo doesn't have to be calculated over and over again.
cpgMIP30k probe set
Two chromosomes enriched in genes differentially expressed between hES/iPS and fibroblasts.
# probes total bps covered CGI covered %CGI covered Chromosome 12 15,998 973,377/924,553 799,818/767,129 82.1%/83.0% Chromosome 20 11,651 654,755/693,920 576,059/550,144 83.0%/84.0%
Additional space for other targets:
- Key genes in maintaining pluripotency or differentiation. 2000bp up/down-stream of the Transcription Start Sites (TSS, based on SwitchGear TSS), ES-Fib-diff: 780 probes
- ENCODE regions, Stanf promoters:
- ENm004: Chr22 151 probes
- ENm005: Chr21 271 probes
- ENm006: ChrX 307 probes
- ENm007: Chr19 276 probes
- ENm010: HOXA cluster 135 probes
- ENm011: 1GF2/H19 188 probes
- ENr123: Chr12 10 probes
- ENr333: Chr20 157 probes
- KH: 293 probes TSS (Hochedlinger: MEG3,SNRPN,CDX1,HAND1,PAX6,FGF7)
Added: ES-Fib-diff 776 (duplicate probes) Total 30,000 Split into three sets: merge chr12 and chr20 probes, sorted by H1/H2 specificity cpgMIP30kV2.1: low score cpgMIP30kV2.2: medium score cpgMIP30kV2.3: high score + other subsets
Agilent probes, V1
- Several considerations in this design include:
- Degenerate bases: I allowed at most two degenerate bases per primer. To convert PCR primers to padlock probes, multiple probes will be designed for one primer pair in order to cover all combinations.
- Gap size: The range of amplicon size is 225-275bp, so the gap size is approximately 185-225bp.
- Amplification adaptors: Version 4; Linker sequences: Version 7
AP1: GTAGACTGGAAGAGCACTGTT V4 AP2: GATCGGATACGCATGAGGCTA V4 Linker: GTTGGAGGCTCATCGTTCCTATTCAGCTGCAGATGTTATCGAGGTCCGAC ------ ---- ------ Mme I Alu I Mme I
- Here is the perl code for primer design. The input file is cpgIslandExt.txt downloaded from UCSC GoldenPath database.
- Conversion of PCR primers to padlock probes: AP1 + revcomp(FP) + linker + RP + AP2. codes.
Some statistics Total probes: 210,498 Total CpG islands: 23,496 Total non-overlapping regions: 12,037,010 bps Total captured sequences: 13,291,608 bps ===CpgMIP28k set=== probes CpG islands Chr6: 9,875 3,028 (1103/1251, 559kb/940kb) Chr20: 6,197 1,930 (695/779, 358kb/613kb) Chr21: 2,619 809 Chr22: 5,358 1,664 (622/716, 310kb/562kb) Chr19: first 3,857
- Probe information file.
- Probe raw sequences.
- ssDNA probes: len=102nt, MW=31KDa.
Column synthesized probes (36)
- Kun:LabNotes/CpgSeq/2007-10-12: probe = revcomp(RP) + linker + FP
- Just realized that there was a mistake in probe design (11-20-2007). I assumed that after bisulfite treatment, the two strands are still complementary, which is not true. The probes have to be designed based on one strand only. If the forward PCR primers contain no "C" and the reverse primers contain no "G", that means the target strand is the forward strand. In that case, the padlock probes should be: revcomp(FP) + linker + RP. Here are the revised perl scripts: first 24 primers; second 12 primers. Here is the new probe file.