samtools mpileup did not call variant

[lchang1101]

I used samtools mpileup and bcftools to call variants in my RNA-Seq data. At one locus, I have 10 reads that are different from the ref sequence (DP4=32,21,5,5), but bcftools did not call this variant.

3 13812321 . T . 47 . DP=65;VDB=0.0190;AF1=0;AC1=0;DP4=32,21,5,5;MQ=20;FQ=-44;PV4=0.73,1,1,1 PL: DP:SP 0:63:1

I tried to change the -p value from the default 0.5 to 0.6, 0.7, 0.8 and 0.9 but still none of them called this variant. I understand that 10 reads in 65 reads is a low fraction, but is there anyway to make bcftools less stringent? I know I can just print out info for every locus and do my own filtering but I was just wondering if there are other parameters of bcftool tat I can modify. Thanks!

[vivek_]

Isn't Mapq=20 rather too low to confidently take the alignment at that position?

[lchang1101]

Thank you for the reply. No, I dont think MQ=20 was the problem. Some of the called variants in my run had MQ as low as 16. Any other thoughts?