Hi,

I want to call variants from a BAM file with bcftools. To use call command, I have to use mpileup command to create a pileup file and use it as input for call. I run default command like below:

bcftools mpileup -f path_to_reference my_file.bam -o my_file.mpileup

The command above gives me this warning:

[mpileup] maximum number of reads per input file set to -d 250

Then I do variant calling with the command below:

bcftools call -m my_file.mpileup -o my_file.vcf

With these settings, a position's genotype is heterozygous (0/1) and depth is 254.

I override max-depth in mpileup with command below:

bcftools mpileup -d 8000 -f path_to_reference my_file.bam -o my_file.mpileup

Same position's genotype becames homozygous to ref (0/0) and depth is 597.

QUAL values are also different.

Why is this happening and which one I can trust?

Thank you!