Athurva Gore/LabNotes/2009-8-25

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Navigation[edit]

ProbeSet Generation[edit]

  • Currently creating TDMR set and CpGSNP set with new no-library-construction protocol
  • This requires a constraint of arms+target = 105
  • This will allow reads of 60-80 bp (after digestion) to cover the entire fragment, greatly improving the capturing protocol (as it seems that library construction is a major source of headaches)

NA12878 False Positives[edit]

  • Noticed that many misses have high number of start/end sites for reads
  • Checked actual read mapping; it seemed that the end of reads was causing the most trouble
    • Many reads had several SNPs in the last few bases
  • After discussing with Dr. Zhang, decided to test mapping and SNP calling using the following protocol.
    • Reads were inspected after mapping to see if any SNPs were present in bases 36-40
    • If any SNPs were present, the entire read was thrown out
    • Hypothesis: Junk is getting ligated onto the ends of reads during library construction
    • If we remove the junk reads, we should be able to greatly improve the false positive rate
  • Remapped and SNP-called the HL006 s_4 (size-selected) and s_5 (not size-selected) sets
    • Filtered out clonal reads
    • Removed reads that had SNPs in locations >= the 36th base
  • Wanted to see if false positive rate went down (though since entire reads are being rejected, coverage will decrease and therefore false negative rate will go up)
  • Results:
    • Stored in /media/TmpStore1/AG_scratch/Exome/NA12878/HL006/noclonal/newtest
    • POST SPECIFIC RESULTS HERE
    • Works extremely well!
    • Number of non-dbSNP calls is reduced to ~100
    • Lose ~1000 hits, but
    • We only lose around 200,000 reads!
    • Works extremely well.
    • Will now apply it to IPS data.

IPS and Cancer[edit]

  • Tested DF-6-9-9 and foreskin fibroblast non-sizeselected reads against each other (HL006, s1 and s3)
  • However, removed clonal reads and reads with SNPs in locations >= 36th base as above
  • Location: /media/TmpStore1/AG_scratch/Exome/DF-6-9-9/HL006/removeBadLig
    • Running mapping/SNP calling scripts now
    • Should reduce number of false positives
  • False positive rate was found to be greatly decreased
    • POST SPECIFIC RESULTS HERE
    • Only a few SNPs not at dbSNP locations compared to before
    • Need to investigate thoroughly, filter out bad calls
  • Now running script to generate variant pairs between S3 and S1; looking for a gain of heterozygosity with particular interest