mpileup file

[Momina]

hi all
I am trying to create a mpileup file so I can get vcf file which I can run on IGV and can use for SNP calling
but i am facing following problems:
i gave following command for mpile up file:
source samtools
samtools mpileup -E -uf reference.fa file.sorted.bam > file.mpileup
it gave output:
[mpileup] 1 samples in 1 input files
<mpileup> Set max per-file depth to 8000
and computer stucks here
when i restart, a mpileup is created
then i try to creat vcf file from that mpileup by giving following command:
source bcftools
bcftools view -cg file.mpileup > file.vcf
error occurs:
Error: Could not parse --min-ac g
can anyone help me in that i am stuck at SNP calling

[GenoMax]

What version of samtools are you using?

[Momina]

samtools-0.1.19

[GenoMax]

Pardon me for the obvious question but are you using your own file names in place of "reference.fa/file.sorted.bam"?

[Momina]

yes
this is what I am commanding:
samtools mpileup -E -uf Triticum_aestivum.IWGSC1.0+popseq.28.dna_sm.genome.fa 606.sorted.bam > 606.mpileup

[GenoMax]

What do you mean by "computer stuck here"? Depending on how large your 606.sorted.bam file it can take a while for the pileup file to be created.

Can you post the output of

$ ls -lh 606*

[Momina]

this is what I ma doing:
source samtools-0.1.19
samtools mpileup -E -uf Triticum_aestivum.IWGSC1.0+popseq.28.dna_sm.genome.fa 604.sorted.bam > 604.mpileup
output:
[mpileup] 1 samples in 1 input files
<mpileup> Set max per-file depth to 8000
after this I press Ctrl C
it starts again this time it does not stuck
but when I write:
source bcftools-1.2
[hussainm@NBI-HPC analysis]$ bcftools view -cg 604.mpileup > 604.vcf
following error occurs
Error: Could not parse --min-ac g

[GenoMax]

It sounds like you are not allowing the first command to complete its work (samtools mpileup). Like I said before it will take some time (could be a few hours if you have a large bam file) for this command to finish. Until that command completes normally you should not start the bcftools process.

[Momina]

[hussainm@NBI-HPC analysis]$ ls -lh 606*
-rwxrwx--- 1 hussainm JIC_a1 12G Aug 21 12:36 606.bam
-rwxrwx--- 1 hussainm JIC_a1 32M Aug 21 14:08 606.sorted.bai
-rwxrwx--- 1 hussainm JIC_a1 7.5G Aug 21 13:30 606.sorted.bam
-rwxrwx--- 1 hussainm JIC_a1 413 Aug 21 15:12 606.sorted_flagstat.txt

[GenoMax]

Looks like you have more than one file that starts with 60* names.

In any case if they are all similar size (e.g. 606.sorted.bam is 7.5G) then it is going to take some time for the mpileup to do its work.

Are you using a job scheduler and/or working on a cluster or is this a standalone server? If this is a standalone server wait for the command prompt to return (do not cancel the job by ctrl+c) after you issue this command:

[hussainm@NBI-HPC analysis]$ samtools mpileup -E -uf Triticum_aestivum.IWGSC1.0+popseq.28.dna_sm.genome.fa 604.sorted.bam > 604.mpileup

[Momina]

ok I will wait now for few hours
and following is the output:
[hussainm@NBI-HPC analysis]$ ls -lh 606*
-rwxrwx--- 1 hussainm JIC_a1 12G Aug 21 12:36 606.bam
-rwxrwx--- 1 hussainm JIC_a1 32M Aug 21 14:08 606.sorted.bai
-rwxrwx--- 1 hussainm JIC_a1 7.5G Aug 21 13:30 606.sorted.bam
-rwxrwx--- 1 hussainm JIC_a1 413 Aug 21 15:12 606.sorted_flagstat.txt

[GenoMax]

Looks like you are using a high performance compute cluster (based on your command prompt). Get some help from your IT support to see if you can submit this job via a job scheduler (so you do not need to keep an interactive session going). If you are running this job on the "head node" that is probably not a good idea anyway.

[Momina]

ya I have 7 files and they all are near 12 -13 GB
and this was a standalone server
now I ma thinking of submitting job
yes I can submit job
and I do that too
I will let you know what there response
following is output of bjobs:
JOBID USER STAT QUEUE FROM_HOST EXEC_HOST JOB_NAME SUBMIT_TIME
62876 hussain RUN NBI-Prod25 v0292.hpccl ncn-768-01. *5.mpileup Aug 26 15:08

[GenoMax]

Great. You can start jobs for other files in parallel so that all 7 get done at the same time. Once those jobs finish go on to the bcftools for the 7 mpileup files.

For future reference you can combine these two processes into one by using unix pipes. See example here: http://samtools.sourceforge.net/mpileup.shtml

[Momina]

Thanku
I saw this before but I didn't get it:
samtools mpileup -uf ref.fa aln1.bam aln2.bam | bcftools view -bvcg - > var.raw.bcf
bcftools view var.raw.bcf | vcfutils.pl varFilter -D100 > var.flt.vcf
aln.bam= these are bam files (probably sorted bam files)?
from where var.raw.bcf comes?
and what is vcfutils.pl??
if you can tell me this with one good example?