Hello all,
An innocuous mpileup gives QUAL of 0 for all bases. 0 shouldn't even be a legal QUAL value (as it is the result of an exponent, according to VCF format documentation). This is the command we're running:
samtools mpileup --VCF -r ChrA_C_glabrata_CBS138:1-100,000 --output-tags DP,DP4 -u -f CBS138.fasta 12.sorted.bam > 12.mpileup.txt
A sample output line:
ChrA_C_glabrata_CBS138 337 . G A,C,T 0 . DP=31;I16=4,11,7,8,530,19116,501,17779,6,6,2,2,298,6778,286,6564;QS=0.5,0.4,0.0666667,0.0333333;VDB=0.523807;SGB=-0.688148;RPB=0.768403;MQB=0.461075;MQSB=0.999074;BQB=0.952765;MQ0F=0.741935 PL:DP:DP4 0,7,3,30,26,5,36,31,9,5:30:4,11,7,8
Thanks!
1 answer
0 is certainly legal, since 10^0 = 1, meaning that there's a 100% chance that the call is wrong. This is unsurprising given that all four possible base changes are apparently present at similar depths and samtools assumes a diploid genome.
There's some filtering that happens before the DP metric is made. This is unlikely to be an mpileup problem per se but rather either a usage problem or simply due to having a bunch of multimappers (otherwise, the depths of the alternate sequences makes no sense). Are you trying to use this for some sort of pooled input?
What do things look like in IGV in this area?
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Another example, that might explain the diploid assignment:
As I said previously, samtools assumes you have a diploid organism. Sites like the one above suggest that either you don't have a haploid organism, you have multimappers, you have incorrect alignments, or you have notable genetic heterogeneity.