Question: samtools mpileup giving QUAL of 0
0
gravatar for blur
5.5 years ago by
blur140
European Union
blur140 wrote:

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!

 

mpileup samtools vcf • 1.7k views
ADD COMMENTlink modified 4.5 years ago by Biostar ♦♦ 20 • written 5.5 years ago by blur140

Another example, that might explain the diploid assignment:

Total count: 95
A      : 0
C      : 67  (71%,     33+,   34- )
G      : 0
T      : 28  (29%,     12+,   16- )
N      : 0
---------------

ADD REPLYlink written 5.5 years ago by blur140

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.

ADD REPLYlink written 5.5 years ago by Devon Ryan97k
0
gravatar for Devon Ryan
5.5 years ago by
Devon Ryan97k
Freiburg, Germany
Devon Ryan97k wrote:

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.

ADD COMMENTlink written 5.5 years ago by Devon Ryan97k

my genome is haploid, and all the SNPs I got look like the line above...

Also, my DP doesn't match the read depth - I opened these files in IGV beforehand, and for example, if IGV has 94 reads, the DP is marked for the same position as 31....

mpileup problem?

ADD REPLYlink written 5.5 years ago by blur140

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?

ADD REPLYlink written 5.5 years ago by Devon Ryan97k

no... this is what worries me...

ADD REPLYlink written 5.5 years ago by blur140

What do things look like in IGV in this area?

ADD REPLYlink written 5.5 years ago by Devon Ryan97k
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