varscan2 reports wrong DP related values

Question

[yingchen]

Hi,

I just found a wired issue with varscan2 and it happened to both version 2.4.4 and 2.4.3. Basically the varscan2 vcf files have wrong DP related values, about 50% off the values reported by IGV or Mutect2 vcf files.

Here is the varscan 2.4.4 vcf for EGFR L858R:

chr7    55259515    .   T   G   .   VarBaseQual ADP=1191;WT=0;HET=1;HOM=0;NC=0 GT:GQ:SDP:DP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR 0/1:27:1191:1191:1179:12:1.01%:1.6693E-3:53:54:1013:166:11:1

Here is gatk Mutect2 vcf for the same variant:

chr7    55259515    .   T   G   .   PASS AC=1;AF=0.500;AN=2;CONTQ=93;ClippingRankSum=0.125;DP=2318;ECNT=2;FS=0.000;GERMQ=93;LikelihoodRankSum=-1.190;MBQ=20,20;MFRL=172,176;MMQ=60,60;MPOS=37;MQ=60.00;MQRankSum=0.000;POPAF=7.30;ROQ=77;ReadPosRankSum=0.086;SEQQ=28;SOR=0.615;STRANDQ=31;TLOD=6.62;UNIQ_ALT_READ_COUNT=26 GT:AD:AF:DP:F1R2:F2R1:SB 0/1:2146,26:9.246e-03:2172:1016,11:1122,14:1029,1117,13,13

When I look at the bam file in IGV, the DP numbers from IGV are almost identical to the values from Mutect2 vcf.

Any explanation?

Thanks,

Ying

Answer 1

[yingchen]

It turned out that it's the issue with samtools mpileup from the varscan pipeline. The version of samtools is 1.9. samtools depth and samtools mpileup report different depths (2325 vs 1193). The depth reported by samtools depth 2325 is similar to depth reported by Mutect2.

samtools view my.bam chr7:55259515-55259515 -c

[W::hts_idx_load2] The index file is older than the data file: myr.bai

7570

samtools depth -d0 -r chr7:55259515-55259515 my.bam

[W::hts_idx_load2] The index file is older than the data file: my.bai

chr7 55259515 2325

samtools mpileup -d0 -r chr7:55259515-55259515 my.bam

[W::hts_idx_load2] The index file is older than the data file: my.bai

[mpileup] 1 samples in 1 input files

chr7 55259515 N 1193 T$T$T$T$t$t$t$t$TTTTTTTTTTTTTTttTTTTTTTTTTTTTT$TTTTTTTTTTTTTTTTTTTTTtttttTTTT$TTTTTTTTTTTTTttTTTTTTTTTTTTTTTTTTTttttTTTTTTTtttttTTTTTTGTTTTTTTTTTTTTTTTTTTTttttTTTTTTTTTTTTTTTTTtTTGTTtTTTTtTTTTttTTTTTTTttTTTTTTTTTTTttttttttTTTTTTTTTTTTtTTTTTTTGTTTTTTTTTTTTTttttTTTTTTTTTTTTTTtttttTTTTTtttttTTTTTTTTTTTTgtttTTTTtttttTTtttGTTttttttTTTTtttttttTTTTTtttTTTTTtttttttttTttttTTTTTTTTtttttttTTtttTtttTTTTTTTTtttTTTttttTTTTTTTTTTTTTTTTTTTTTTTTTTtttTTTTTTTTTTTTTTTTTTTttTTTTTttTTTTTTTTTTTTTTTTTTTttttttttTTTTttttTTTTTTTGTtTTTTTTTTTTTTTTTTTTTTTTTTTtttttTTTTTTTTTTTTTTTtTTTTTTtTTTTTTTTTTTTTttTTTTTTTTTtTTTTTTTTTTttTTTttTtTTTTTTTTTTTTTTTTTTTTTTTTTTTttttttttTTTTTTTTTTTTTTTTTTTTTTTGTTTTTTTTTTTTTTTTTTTTTTTTTTtTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTTTTTTTTTTTTTTTTTtTTTTTTTTTTTTTTTTTTTTtTTTtTTTTTTTTTTTTTTTTTTTTTTTTTTTTTtTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTtTTTTTTTTTTTTTTtTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT^]T ]]]]????]]]]]]]]]]]]]]??]]]SS]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]?????]]]]]]]S]]]]]]]]]??]]]]]]]]]]]]]]]]]]]????]]]]]]]?????]]]]]]]]]]]]]]]]]]]]]]]]]]]????]]]]]]]]]]]]]]]]]?]]]]]?]]]]?]]]]??]]]]]]]??]]]]]]]]]]]????????]]]]]]]]]]]]?]]]]]]]]]]]]]]]]]]]]]????]]]]]]]]]]]]]]?????]]]]]?????]]]]]]]]]]]]????]]]]?????]]???]]]??????]]]]9??????]]]]]???]]]]S?????????]????]]]]]]]]???????]]???]???]]]]]]]]???]]I????]]]]]]]]]]]]]]]]]]]]]]]]]]???]]]]]]]]]]]]]]]]]]]??]]]]]??]]]]]]]]]]]]]]]]]]]????????]]]]????]]]]]]]]]?]]]]]]]]]]]]]]]]]]]]]]]]]?????]]]]]]]I]]]]]]]?]]]]]]?]]]]]]]]]]]]]??]]]]]]]]]?]]]]]]]]]]??]]]??]?]]]]]]]]]]]]]]]]]]]]]I]]]]]????????]S]]]]]]]]]]]]]]]]]]]]]]]]]]]]I]]]]]]]]]]]]]]]]]]]?]]]]]]]]]]]]]]]]]]]]]]]]S]]]]]]]]]]]]]]]]]]]]]]]]?]]]]]]]]]]]]]]]]]]]]?]]]??]]]]]]]]]]]]]]]]]]]]]]]]]]]]9]]]]]]]]]]]]]]]]]]]]]?]]]]]]]]]]]]]]]]]]]]]]9]]]]?]]]]]]]]]]]]]]]]]]]]]]]]]]?]]]]]]]]]]]]]]?]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]]?]]]]]]]]]]]]]]]]]]]]]]]]]]?]]]]]]]]]]]]]]]]]]]]]]]]]]]]]S]]]]]?]]]?]]]]]]]]]]]]]]]]?]]]]]S]]]]]]]]]]]]]]?]]]]]]]?]]]]]]]]?]]?]]?]]]]]]]]?]]]]?]]]]]?]]??]]]]]?]?]]?]?]?]?]?]]]]?]]?]?]??]]]]]]]]]????]??]?]S]]]???]]?]S?]???]]]????????]?]]]?]?]?]?]?]???]???]???]]???????]]?]?]]?

Any suggestion?

Thanks a lot!

Ying

Answer 2

[steve]

In my Varscan2 .vcf header it describes the DP field like this:

##FORMAT=<ID=DP,Number=1,Type=Integer,Description="Quality Read Depth of bases with Phred score >= 15">

Whereas for MuTect2, my .vcf header says this:

        ##FORMAT=<ID=DP,Number=1,Type=Integer,Description="Approximate read depth (reads with MQ=255 or with bad mates are filtered)">

So perhaps Varscan2 is applying more filtering to the reads it is counting?