VCF file - 'X' bases in the ALT position and QUAL = 0 . Why ?

[north_zeb]

I am completely new to samtools pileup and bcftools. I hope to stumble here on someone more knowledgeable than me !

So i have two alignments and a reference genome and here is how i use the samtools command:

samtools mpileup -d 200 -D -B -f ../hg19_bt2/hg19-bt2-index.fa -b bamlist.txt -u | bcftools view - -v -c -g > variants.vcf

By doing this i would like to call variants only and call genotypes as well.

The command is still running but when i am having a look at my result file i don't quite like what i see:

#CHROM  POS     ID      REF     ALT     QUAL    FILTER  INFO    FORMAT  gcancer.srtd.reh.ddup.bam       normal.srtd.reh.ddup.bam
chr1    10001   .       T       C,X     0       .       DP=3;I16=2,0,0,1,75,2813,17,289,1,1,1,1,0,0,0,0 PL:DP   0,0,0,0,0,0:0   0,3,3,6,6,4:3
chr1    10002   .       A       X       0       .       DP=4;I16=3,1,0,0,133,4657,0,0,2,2,0,0,3,3,0,0   PL:DP   0,0,0:0 0,12,13:4
chr1    10003   .       A       X       0       .       DP=5;I16=4,0,0,0,151,5701,0,0,1,1,0,0,5,9,0,0   PL:DP   0,3,4:1 0,9,9:3
chr1    10004   .       C       X       0       .       DP=5;I16=4,0,0,0,150,5626,0,0,1,1,0,0,9,23,0,0  PL:DP   0,3,4:1 0,9,9:3
chr1    10005   .       C       X       0       .       DP=5;I16=4,1,0,0,180,6542,0,0,2,2,0,0,17,61,0,0 PL:DP   0,3,4:1 0,12,13:4

-> First of all, i see that each base per chr is shown. Haven't i asked to get the potential variant sites only ? (-v)
-> I don't get these 'X's at the ALT position. If there is no variant, why just not omit the position?
-> Then, i'm absolutely lost why my QUAL is 0. All the time, everywhere in this vcf file. What am i missing ?
-> Then, in the genotype fields, i get these comma separated PL which i read is (PL : the phred-scaled genotype likelihoods rounded to the closest integer (and otherwise defined precisely as the GL field) (Integers)) but why there are several and not just one for each of the two samples?
-> And then why the DP in INFO does not equal to the DP in the genotypes ? For example, in the third row, DP=5 in INFO but it is 1 and 3 respectively for each of the 2 samples ?

Thank you in advance guys !

[danwiththeplan]

I am also getting an X in a variant position with QUAL=0, this also causes the VCF file to fail when loading into IGV... can anyone explain this?

[sunnycqcn]

Did you find why it is?
I have same question.

[sunnycqcn]

Hi Sir,
Did you find why it is now? I have same problem.
Could you tell me how to do?
Thanks,

I am completely new to samtools pileup and bcftools. I hope to stumble here on someone more knowledgeable than me !

So i have two alignments and a reference genome and here is how i use the samtools command:

samtools mpileup -d 200 -D -B -f ../hg19_bt2/hg19-bt2-index.fa -b bamlist.txt -u | bcftools view - -v -c -g > variants.vcf

By doing this i would like to call variants only and call genotypes as well.

The command is still running but when i am having a look at my result file i don't quite like what i see:

#CHROM  POS     ID      REF     ALT     QUAL    FILTER  INFO    FORMAT  gcancer.srtd.reh.ddup.bam       normal.srtd.reh.ddup.bam
chr1    10001   .       T       C,X     0       .       DP=3;I16=2,0,0,1,75,2813,17,289,1,1,1,1,0,0,0,0 PL:DP   0,0,0,0,0,0:0   0,3,3,6,6,4:3
chr1    10002   .       A       X       0       .       DP=4;I16=3,1,0,0,133,4657,0,0,2,2,0,0,3,3,0,0   PL:DP   0,0,0:0 0,12,13:4
chr1    10003   .       A       X       0       .       DP=5;I16=4,0,0,0,151,5701,0,0,1,1,0,0,5,9,0,0   PL:DP   0,3,4:1 0,9,9:3
chr1    10004   .       C       X       0       .       DP=5;I16=4,0,0,0,150,5626,0,0,1,1,0,0,9,23,0,0  PL:DP   0,3,4:1 0,9,9:3
chr1    10005   .       C       X       0       .       DP=5;I16=4,1,0,0,180,6542,0,0,2,2,0,0,17,61,0,0 PL:DP   0,3,4:1 0,12,13:4

-> First of all, i see that each base per chr is shown. Haven't i asked to get the potential variant sites only ? (-v)
-> I don't get these 'X's at the ALT position. If there is no variant, why just not omit the position?
-> Then, i'm absolutely lost why my QUAL is 0. All the time, everywhere in this vcf file. What am i missing ?
-> Then, in the genotype fields, i get these comma separated PL which i read is (PL : the phred-scaled genotype likelihoods rounded to the closest integer (and otherwise defined precisely as the GL field) (Integers)) but why there are several and not just one for each of the two samples?
-> And then why the DP in INFO does not equal to the DP in the genotypes ? For example, in the third row, DP=5 in INFO but it is 1 and 3 respectively for each of the 2 samples ?

Thank you in advance guys !