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 |