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Kun:LabNotes/ExonomeSeq/2009-11-15
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==Analysis of iPS exome sequencing data== *Data used: **Libraries were constructed with the 1-adaptor protocol. **DF6/Foreskin: HL022, two lanes per line, captured with probe set #1-7; **CV-iPS/CV-fibroblast: ***HL020, two lanes per line, captured with probe set #8-9; ***HL022, three lanes for CV-iPS, captured with probe set #1-7; *Read mapping and variant calling. **Reads that contain potential H1/H2 capturing arms are identified and removed by mapping to a database that contains H1/H2 with 20bp flanking sequences. **The remaining reads were mapped, down-sampled and fed to Samtools. **This is the current mapping script: [[media:variantCallerBowtieSam.txt|variantCallerBowtieSam.pl]]. **Potential mutations were identified by comparing two pileup files ([[media:pileup2variantsPair.txt| pileup2variantsPair.pl]]); **To be considered as a candidate mutation, I required that no single read contains the second allele in the reference sample. *Results: ** 4 candidates of single nucleotide changes (SNCs) found in CV-iPS:[[media:CV-iPS_vs_fibro_set8-9_HL020.snc.xlsx|CV-iPS_vs_fibro_set8-9_HL020]]; ** 5 candidates of SNCs found in DF6-9-9:[[media:DF6_Foreskin_HL022_snc.xlsx|DF6_Foreskin_HL022]]; ** Nine pairs of PCR primers were designed for these variants. However, ZNF708-K259R will be excluded in the first validation experiment because the flanking sequence has many highly similar homologs on the same chromosome. {| {{table}} | align="center" style="background:#f0f0f0;"|'''PrimerID''' | align="center" style="background:#f0f0f0;"|'''LP''' | align="center" style="background:#f0f0f0;"|'''PrimerID''' | align="center" style="background:#f0f0f0;"|'''RP''' | align="center" style="background:#f0f0f0;"|'''Amplicon''' |- | TRIM25-N534K-LP||GTGGCACCAGCAGAAAATAC||TRIM25-N534K-RP||TCCCCAGAGGTTCACATACT||>chr17:52324085+52324480 396bp |- | SDR16C5-D139Y-LP||GTGAGCCACCTGGAGTATTT||SDR16C5-D139Y-RP||AAAGAAGTCGGCGATGTTTC||>chr8:57386991+57387395 405bp |- | NTRK3-L585R-LP||ACAATGCCTAGAGCTTCCAA||NTRK3-L585R-RP||CTGCAGCAAAATGGAGTGTT||>chr15:86323500+86323903 404bp |- | DAPL1-P18P-LP||GTCTCCTGCAGACATCTACC||DAPL1-P18P-RP||CAGAGAAGCTTCCGTCTGTC||>chr2:159359887+159360285 399bp |- | ZZZ3-K214R-LP||CTTACCCTCCACTTTTGCAC||ZZZ3-K214R-RP||ACGACTCAGGTATTGTATGTC||>chr1:77816893+77817200 308bp |- | PPP1R2-P97R-LP||CAGTATCCCAAGTGCAGGTA||PPP1R2-P97R-RP||CCCTTGTTTTTGTGGCTGAA||>chr3:196732652+196733047 396bp |- | ITCH-T556A-LP||ACCCATGGAAGCAAGAAGAA||ITCH-T556A-RP||CAAAAGCGGGTCCTAAGCTA||>chr20:32522599+32523040 442bp |- | NEK5-G23E-LP||TGGTTGCTGTTTGGACAATG||NEK5-G23E-RP||GCTGGGAGGAAAGTTTTAGC||>chr13:51599342+51599754 413bp |}
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