VCF file - no ALT but DP4 alternative exist

[Rachelly]

Hi all,

I'm using samtools to find variants in my resequencing data, and I've noticed something weird:

After running the following command:

samtools mpileup -L 10000 -d 10000 -ugEf <my_genome> <my sorted BAM> | bcftools view -g - > samp.raw.vcf

I got a line in the VCF file that doesn't make sense: It said that there is no variant (ALT=".", AF1="0"), but there are alternative alleles, as can be seen in the DP4 attributes. I also see it clearly in the IGV when viewing the BAM file.

genome     197469  .       T       .       72      .       DP=666;VDB=0.0469;AF1=0;AC1=0;DP4=258,220,75,110;MQ=33;FQ=-69;PV4=0.0024,1,3.4e-120,1   PL      0

And the weirdest thing is that when I ran the same command on a different sample, the output was ok (both samples contain the same SNP):

genome     197469  .       T       C       27      .       DP=666;VDB=0.0469;AF1=0.5;AC1=1;DP4=258,220,75,110;MQ=33;FQ=30;PV4=0.0024,1,3.4e-120,1  GT:PL:GQ        0/1:57,0,255:60

Anybody knows what's going on? I've never seen this behavior before with Samtools.

Thanks,
Rachelly.

[swbarnes2]

What does the pileup look like at that locus?

[Rachelly]

The pileups looks the same..

The pileup of both samples looks exactly the same. See the attached image from IGV.



I found more examples for this behavior in these samples, where they look the same in IGV but their VCF files look different:

genome     256638  .       C       .       82      .       DP=702;VDB=0.0188;AF1=0;AC1=0;DP4=319,217,94,70;MQ=33;FQ=-79;PV4=0.65,1,1.3e-98,1       PL      0
genome     256638  .       C       A       4.77    .       DP=702;VDB=0.0188;AF1=0.4999;AC1=1;DP4=319,217,94,70;MQ=33;FQ=6.99;PV4=0.65,1,1.3e-98,1 GT:PL:GQ        0/1:33,0,255:33

genome     217418  .       A       .       15.1    .       DP=940;VDB=0.0028;AF1=0.492;AC1=1;DP4=316,372,116,121;MQ=33;FQ=-12.3;PV4=0.45,2.7e-05,9.7e-135,4.6e-10  PL      15
genome     217418  .       A       T       5.46    .       DP=940;VDB=0.0028;AF1=0.4999;AC1=1;DP4=316,372,116,121;MQ=33;FQ=7.8;PV4=0.45,2.7e-05,9.7e-135,4.6e-10   GT:PL:GQ        0/1:34,0,255:34