varscan2 somatic sip

Question

[fabbri.marco]

I have used varscan2 and the results below are not clear to me.I compared a tumour to a normal and I am interested in somatic mutation. The following snp is predicted as germline and I expected as somatic but it is not clear to me the reason, Any suggestions? Thanks Marco

##fileformat=VCFv4.1
##source=VarScan2##INFO=<ID=DP,Number=1,Type=Integer,Description="Total depth of quality bases">
##INFO=<ID=SOMATIC,Number=0,Type=Flag,Description="Indicates if record is a somatic mutation">
##INFO=<ID=SS,Number=1,Type=String,Description="Somatic status of variant (0=Reference,1=Germline,2=Somatic,3=LOH, or 5=Unknown)">
##INFO=<ID=SSC,Number=1,Type=String,Description="Somatic score in Phred scale (0-255) derived from somatic p-value">
##INFO=<ID=GPV,Number=1,Type=Float,Description="Fisher's Exact Test P-value of tumor+normal versus no variant for Germline calls">
##INFO=<ID=SPV,Number=1,Type=Float,Description="Fisher's Exact Test P-value of tumor versus normal for Somatic/LOH calls">
##FILTER=<ID=str10,Description="Less than 10% or more than 90% of variant supporting reads on one strand">
##FILTER=<ID=indelError,Description="Likely artifact due to indel reads at this position">
##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype">
##FORMAT=<ID=GQ,Number=1,Type=Integer,Description="Genotype Quality">
##FORMAT=<ID=DP,Number=1,Type=Integer,Description="Read Depth">
##FORMAT=<ID=RD,Number=1,Type=Integer,Description="Depth of reference-supporting bases (reads1)">
##FORMAT=<ID=AD,Number=1,Type=Integer,Description="Depth of variant-supporting bases (reads2)">
##FORMAT=<ID=FREQ,Number=1,Type=String,Description="Variant allele frequency">
##FORMAT=<ID=DP4,Number=1,Type=String,Description="Strand read counts: r
#CHROM  POS ID  REF ALT QUAL    FILTER  INFO    FORMAT  NORMAL  TUMOR
chr12   49420661    .   G   A   .   PASS    DP=3204;SS=1;SSC=255;GPV=1E0;SPV=1.5981E-225    GT:GQ:DP:RD:AD:FREQ:DP4 0/1:.:1843:1835:2:0.11%:1595,240,2,0    0/1:.:1361:828:532:39.12%:740,88,475,57

[fabbri.marco]

I think that I will change my setting, since this is in my opinion is a somatic mutation, there only 2 reads in the normal sample (lymphoma). I check how to set varscan option in a way that this should be predicted as somatic. What do you think? Marco

[WouterDeCoster]

Please use ADD COMMENT or ADD REPLY to answer to previous reactions, as such this thread remains logically structured and easy to follow. I have now moved your post but as you can see it's not optimal. Adding an answer should only be used for providing a solution to the question asked.

Answer 1

[WouterDeCoster]

The SNP is found in both your tumor and your normal sample (both have a 0/1 genotype). Therefore it's not somatic but germline.

[ATpoint]

The allel frequency in the normal is 0.11%, but 39% in the tumor. This could be due to a tumor contamination of the normal control.

[WouterDeCoster]

That's a good point. Although I would expect that if contamination happens it's the other way around. But we don't know how the samples were collected and which type of tumor is studied.

[ATpoint]

It is a lymphoma sample so probably tumor is a lymph node biopsy, and normal is PBMC . Would not be too uncommon if some malignant B cells escaped the lymphatic system and started circulating.

[WouterDeCoster]

Right, sounds reasonable.

[fabbri.marco]

Yes the sounds reasonable, I have to check the settings in varscan. Probably I have to allow to define Germaine above a percentage or number of reads. Any suggestions?

[ATpoint]

There is the --max-normal-freq flag in ./VarScan2 processSomatic, default is 0.05 so 5% as maximum frequency for normals. Did you reduce that to 1%? If so, try with the defaults. I think this makes biological sense to exactly exclude these kinds of situations you experience right now.