SNV ignored by bcftools

[behrj]

Hi everyone,

I am currently using bcftools to call variants and saw some strange behavior. It would be perfect if someone could explain this to me, because I did not find any
hint from reading the paper and the bcftools documentation. The question is, looking at the mpileup I can see that roughly one third of the reads has a
C instead of a T. With a coverage of several hundred, this should not be a problem to call. However, if I do not use the option -C50 this variant is not called by bcftools. On the other hand I do not want to use -C50, because then I miss variants that are close to other variants in the genome.

Any idea?

echo mpileup
$samtools mpileup $fn_bam -f $fn_genome -E -r $chr:$((pos-$off))-$((pos+$off))
echo "mpileup | bcftools"
$samtools mpileup $fn_bam -f $fn_genome -E -r $chr:$((pos-$off))-$((pos+$off)) -u | $bcftools view -cg '-' | grep -v "^#"
echo "mpileup -C50 | bcftools"
$samtools mpileup $fn_bam -C50 -f $fn_genome -E -r $chr:$((pos-$off))-$((pos+$off)) -u | $bcftools view -cg '-' | grep -v "^#"

The result is the following (I show just some part of the pileup columns, the coverage is high):

mpileup
1       88212463        T       293     ,$,,,.,..,.,.,..,,,,,,,....,,...,......,.,..,...,,....,,.......,,.,....,..,,,,..,...,,,......,,..,...,....,.,,,.,,,,.,.,.......,,.....,,..,,,..,.......,.,,,,..,...,......,,,...,,.,.......,,.,,..,,...
1       88212464        T       293     ,$,$,.,..,.,.,..,,,,,,,....,,...,......,.,..,...,,....,,.......,,.,....,...,,,..,...,,,.....,,..,...,....,.,,,.,,,,.,.,.......,,.....,,..,,,..,.......,.,,,,..,...,......,,,...,,.,.......,,.,,..,,...,
1       88212465        T       296     ,$.,$..,.,C,.C,,,,,,,....,cC..cCC..C.,.,..,..,c...C,,.....C.,,.c....,..Ccc,,.C,..C,,c...C..,cC.,C..,....c.,,,.,,,,C,.c...C..C,,.....,cC.,c,..cCC...C.,.,,,c..,...,.CCC..,,c...c,C,.C.C.Cc,.,,.C,,..C,,.
1       88212466        C       299     .$.$.$,.,.,..,,,,,,,....,,...,......,.,..,...,,....,,.......,,.,....,...,,,..,...,,,.....,,..,...,....,.,,,.,,,,,.,.......,,.....,,..,,,..,.......,.,,,,..,...,......,,,...,,.,.......,,.,,..,,...,,..,
1       88212467        C       298     ,$,.,..,,,,,,,....,,...,......,.,..,...,,....,,.......,,.,....,...,,,..,...,,,......,,..,...,....,.,,,.,,,,.,.,.......,,.....,,..,,,..,.......,.,,,,.,...,......,,,...,,.,......,,.,,..,,...,,..,,....,
mpileup | bcftools
1       88212463        .       T       .       283     .       DP=302;AF1=0;AC1=0;DP4=184,109,0,0;MQ=20;FQ=-282        PL      0
1       88212464        .       T       .       283     .       DP=306;AF1=0;AC1=0;DP4=186,107,0,0;MQ=20;FQ=-282        PL      0
1       88212465        .       T       .       144     .       DP=313;VDB=6.428646e-02;RPB=7.216996e-01;AF1=0;AC1=0;DP4=146,86,42,22;MQ=20;FQ=-141;PV4=0.77,0.43,1,1   PL      0
1       88212466        .       C       .       283     .       DP=314;AF1=0;AC1=0;DP4=193,106,0,0;MQ=20;FQ=-282        PL      0
1       88212467        .       C       .       283     .       DP=316;AF1=0;AC1=0;DP4=191,107,0,0;MQ=20;FQ=-282        PL      0
mpileup -C50  | bcftools
1       88212463        .       T       .       283     .       DP=292;AF1=0;AC1=0;DP4=178,105,0,0;MQ=43;FQ=-282        PL      0
1       88212464        .       T       .       283     .       DP=295;AF1=0;AC1=0;DP4=179,103,0,0;MQ=43;FQ=-282        PL      0
1       88212465        .       T       C       60      .       DP=299;VDB=6.578340e-03;RPB=-5.410734e-01;AF1=0.5;AC1=1;DP4=141,83,38,20;MQ=43;FQ=63;PV4=0.76,0.34,0.0003,1     GT:PL:GQ        0/1:90,0,255:93
1       88212466        .       C       .       283     .       DP=299;AF1=0;AC1=0;DP4=183,101,0,0;MQ=43;FQ=-282        PL      0
1       88212467        .       C       .       283     .       DP=299;AF1=0;AC1=0;DP4=179,102,0,0;MQ=43;FQ=-282        PL      0