Problems with samtools mpileup

Question

[mangfu100]

Hi all.

I am using mpileup to extract some sequencing information. However, when I tried to run mpileup with below command

samtools mpileup -C 50 -vu -t DP -t DV -t DPR -t DP4 -f human_g1k_v37.fasta -l ms_TAR22_mpileup.bed TAR22_N_Filtered_Sorted_Makrdup_Readgroup_Realigned_Recal.bam

The results are nothing except head file of VCF.

I think that the problems occur due to the -l option because when I removed -l options, it works well but the file size was very large to handle. When I chose -l option and run it, as I expected, then it failed to generate sequencing information related to -l files. I don't know why mpileup command doesn't output anything but its head even though I followed the manual and ran with the latest samtools version(1.2).

For your reference, here is the data inside my bed files.

22      24829532        24829532        ADORA2A
18      12449779        12449779        SPIRE1
12      123005948       123005948       RSRC2
12      25398281        25398281        KRAS
12      22354743        22354743        ST8SIA1
7       93055822        93055822        CALCR
2       170374777       170374777       KLHL41
1       158532510       158532510       OR6P1
1       79002163        79002163        PTGFR

Help me to get it!

Answer 1

[Sean Davis]

BED files are 0-based, half-open. What that means is that a SNV location needs to have a difference of 1 between the start and end location. In the case of your BED file, the locations appear to be in 1-based, closed coordinates. So, I suspect that mpileup is not using the BED file as you think. Try subtracting 1 from each element in column 2. Finally, make sure that the chromosome names match (including the "chr" or lack thereof).