calling Heterozygous SNPs with samtools mpileup

[egatti]

Dear all,

I am trying to use samtools mpileup in order to call the variants in my bam file.
I know that in my sample there are some homozygous and heterozygous SNPs. But samtools seems to not call a lot of mine heterozygous SNPs.
Maybe I did not get an important point of the mpileup command, but looking in the man page of sourceforge of samtools I was not able to find any solutions to my problem.
Does anyone can help me?

Here my command:
samtools mpileup -ug -f fasta.fa aln.bam | bcftools view -bvcg - > var.raw.bcf
bcftools view var.raw.bcf | vcfutils.pl varFilter -D1000000 > var.flt.vcf

Thank you so much

Elena

[swbarnes2]

I think the command is fine. First obvious thing; look at the pileup at the loci where you expect SNPs, and see what they look like.

Second thing, try redoing the command line with -Bug or -Eug. The BAQ calculations sometimes will degrade the quality at real SNPs, such that they can't be called. I've seen this happen with sanger verified SNPs

Also, the samtools manual recommends -C50 in the mpileup command if you are using bwa to align.