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==Design of the CES27K9bp probe set (Aug. 2008)== *This is the second probe set that has a gap larger than 1bp. The major differences between CES27K9bp and CES27K are: **The gap size is 9bp, and the SNPs are 2bp away from the ligation arms (H1) and 6bp away from the extension arms (H2). The reason to place the SNPs further way from the extension arms is to increase the annealing stability of the extension arm with the extension of 6bp before the SNPs are filled. Hopefully this will reduce the allelic difference of circularization efficiency for the probes. **The annealing temperature was increase to ~65C for the ligation arms and ~53C for the extension arms. *Procedure for probe design: **Map common SNPs (MAF>=0.05) to RefSeq genes: [[Media:snp2RefGeneV2.txt|codes]]; **Extract 50bp upstream/downstream sequences for the SNPs from the transcriptome: [[Media:getSnpcDNAFlankingSeq.txt|codes]]; **Design capturing arms for each SNPs, check for the uniqueness of each captured target: [[Media:CircPlex_bigGapsV2.txt|codes]]; **Convert the sequences for capturing arms into Agilent oligo libraries: [[Media:p2MIP_v9.txt|codes]] **Find the MAF and the distance to splicing junctions for each SNP (should have been included earlier): [[Media:annoSNPs.txt|codes]]; **Select a subset of probes in Excel based on: ***Tm(H1): 60-72C; Tm(H2): 50-62C; ***Distance to splicing junctions: at least 25bp; ***MAF: at least 0.075 **MW: probe=33KD; Solexa library=105KD (171bp) *[[Media:snp_refseq_coding_9bp.probes.anno.xlsx|Probe annotation file]]. *[[Media:CES27k9bp.txt|raw probe sequences]]. *[[Media:CES27k9bp_2008-10-11-read-counts.txt|Capturing efficiencies based on end-sequencing.]] [[Media:CES27k9bp_capturing_efficiency.txt|A more complete data set.]]
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