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===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?[[Media:getCpgProbes.txt|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 [[Media:bisCvt.txt|bisulfite converted]] genome were [[Media:oligoSamp.txt|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 #Genes suggested by the Hochedlinger lab (MEG3,SNRPN,CDX1,HAND1,PAX6,FGF7) KH: 293 probes. [[Kun:LabNotes/CpgSeq/Cpg30k_selected_genes_list|Gene list of #1 & #2.]] #ENCODE regions, Stanf promoters: ENm004: Chr22 151 probes chr22:30,133,954-31,833,953 ENm005: Chr21 271 probes chr21:32,668,237-34,364,221 ENm006: ChrX 307 probes chrX:152,767,492-154,063,081 ENm007: Chr19 276 probes chr19:59,023,585-60,024,460 ENm010: HOXA cluster 135 probes chr7:26,924,046-27,424,045 ENm011: 1GF2/H19 188 probes chr11:1,699,992-2,306,039 ENr123: Chr12 10 probes chr12:38,626,477-39,126,476 ENr333: Chr20 157 probes chr20:33,304,929-33,804,928 Added: ES-Fib-diff 776 (duplicate probes) Total 30,000 Split into three sets: merge chr12 and chr20 probes, sorted by H1/H2 specificity [[Media:cpgMIP30kV2.1b.txt|cpgMIP30kV2.1: low score]] [[Media:cpgMIP30kV2.2b.txt|cpgMIP30kV2.2: medium score]] [[Media:cpgMIP30kV2.3b.txt|cpgMIP30kV2.3: high score + other subsets]] [[Media:cpgMIP30k_probeInfo.txt|Probe information]] [[Media:cpgMIP30k_2008-06-12-read-counts.txt|Capturing Efficiencies based on end-sequencing.]] ====Additional probes for normalizing the cpgMIP30k set==== *A cpgMIP30k captured library was end-sequenced on 06/12/08, 10364/10582 targets showed up at least once. So I sorted all 10582 probe templates in the order from low abundance to high (abundance for the ones did not showed up were considered 0). These templates were converted to probes with probe2padlockCpgLibV2.pl, and split into four sets (the last one not synthesized). #cpgMIP30kNormL: 4677 probes, abundance: 0-10; the remaining space was filled with 5323 CES22k set on the low end of the distribution on gDNA capture (based on Hues62/63 data on 06/12/08). #cpgMIP30kNormM: 9,999 probes (one included twice), abundance: 11-80 #cpgMIP30kNormH: 10,000 probes, abundance: 80-881
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