Kun:LabNotes/ExonomeSeq/2009-11-15

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Analysis of iPS exome sequencing data[edit]

  • 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: variantCallerBowtieSam.pl.
    • Potential mutations were identified by comparing two pileup files ( 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:CV-iPS_vs_fibro_set8-9_HL020;
    • 5 candidates of SNCs found in DF6-9-9: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.
PrimerID LP PrimerID RP 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