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=====Second round (for missing ccdsExons)===== **Some exons were missing because they were on chrN_random based on the refGene annotation. Athurva has generated a list of ccdsExon and ccdsExonLink based on ccdsGenes downloaded from UCSC. I used this [[Media: getMissingCcdsExon.txt|perl script]] to map all probes in the Jan09 set to ccdsExonLink, and generated a list of the [[Media: ccdsExon_not_designed.txt|missing ccdsExons]] (17,111 in total). **I used [[Media: getExonProbes1stRound.txt|getExonProbe1stRound.pl]] to do the first pass of probe design on the list of missing ccdsExons. In this round of design, SNPs or repetitive sequences are not allowed in H1/H2. **Then I ran [[Media: getExonProbes2ndRound.txt|getExonProbe2ndRound.pl]] on the remaining 5578 ccdsExons. SNPs or repetitive seqeuences are allowed outside the 12bp terminal sequences of H1/H2. **For the 4020 exons that no probes could be designed in the previous two passes, I ran [[Media: getExonProbes3rdRound.txt|getExonProbe3rdRound.pl]] with even less stringent criteria: ***Wider Tm range (48-75C); ***Target length extended to 225bp; ***Allow for more than one probe for small exons. ***After this third design, only 93 exons were still missing. **A total of 33235 probes were designed for the missing ccdsExons after three rounds. **27150/33235 probes capture unique targets (no homologous sequence with >=95% similarity) **Combining the 33,235 probes with the 258,861 probes ordered in the Jan09 set, a total of 27.3Mb (91%) CCDS exons were covered. 89% CCDS exons were covered by 27,150+258,861 probes. **[Feb-16-2009] Just realized that the ccdsExon set generated by Athurva contains 1,505 redundant exons(1679 probes), which were alternative splicing isoforms of RefSeq genes. After these redundant ones were removed (or the shorter ones combined to longer ones), the total CCDS set contains 163,641 unique exons. Including 685 miRNAs, we have a total of [[Media:ccdsExon_unique_miRNA.txt|164,326 targets]]. Some of the adjacent exons are separated by small introns of less than 200bp in size. Since we are including 80-120bp flanking sequences on both sides of an exon for probe design anyway, I decided to merge these adjacent exons if the intron size is less than 200bp. After the merging, I got [[Media:ccdsExon_unique_miRNA_200bp.merge.txt|145,532 targets]]. ***The list of missing exons was reduced from 17,111 to 15,744: [[Media: ccdsExon_not_designed_unique.txt|missing unique ccdsExons]] ***The number of probes for these unique exons was reduced to 22,271, among which 16,566 capture unique targets.
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