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  • mpileup file

    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

  • #2
    What version of samtools are you using?

    Comment


    • #3
      samtools-0.1.19

      Comment


      • #4
        Pardon me for the obvious question but are you using your own file names in place of "reference.fa/file.sorted.bam"?

        Comment


        • #5
          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

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          • #6
            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

            Code:
            $ ls -lh 606*

            Comment


            • #7
              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

              Comment


              • #8
                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.

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                • #9
                  [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

                  Comment


                  • #10
                    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:

                    Code:
                    [hussainm@NBI-HPC analysis]$ samtools mpileup -E -uf Triticum_aestivum.IWGSC1.0+popseq.28.dna_sm.genome.fa 604.sorted.bam > 604.mpileup
                    Last edited by GenoMax; 08-26-2015, 06:06 AM.

                    Comment


                    • #11
                      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

                      Comment


                      • #12
                        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.

                        Comment


                        • #13
                          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

                          Comment


                          • #14
                            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

                            Comment


                            • #15
                              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?

                              Comment

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