As sort of a pilot study, we used Solid 5500 (Agilent SureSelect Exome) to sequence one
tumor tissue and one matched normal tissue from the same patient.
I used the vendor-provided LifeScope Genome Analysis Software to align the sequence and did the SNP callings. The initial SNP call has about 50,000 entries.
For every SNP called in tumor, I aligned the Consensus Call of the same base position in the matched normal tissue, and then filtered out ones where the same base position in matched normal showed evidence of SNP.
I also filtered out those in the dbSNP.
We tested a small number of those potentially tumor-specific SNPs in Sanger sequencing, picking out ones with high coverage. 90% of them turned out to be sequencing error. For the other 10%, the same variant is also found in the matched normal.
Well obviously, my noob approach didn't work.
I picked out some SNPs to test because the bases had high coverage (although the reference vs. novel call is hardly 50-50, for most it's more like 80-20), although after the fact I do notice that many of them had pretty low Quality Score (QS = 20 or less).
So anyway, not too many discussions about LifeScope, do you guys actually recommend some different software for such analysis? Or is there something I'm totally missing here?
Thanks in advance.
tumor tissue and one matched normal tissue from the same patient.
I used the vendor-provided LifeScope Genome Analysis Software to align the sequence and did the SNP callings. The initial SNP call has about 50,000 entries.
For every SNP called in tumor, I aligned the Consensus Call of the same base position in the matched normal tissue, and then filtered out ones where the same base position in matched normal showed evidence of SNP.
I also filtered out those in the dbSNP.
We tested a small number of those potentially tumor-specific SNPs in Sanger sequencing, picking out ones with high coverage. 90% of them turned out to be sequencing error. For the other 10%, the same variant is also found in the matched normal.
Well obviously, my noob approach didn't work.
I picked out some SNPs to test because the bases had high coverage (although the reference vs. novel call is hardly 50-50, for most it's more like 80-20), although after the fact I do notice that many of them had pretty low Quality Score (QS = 20 or less).
So anyway, not too many discussions about LifeScope, do you guys actually recommend some different software for such analysis? Or is there something I'm totally missing here?
Thanks in advance.
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