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  • Many Ns in samtools mpileup

    Hi,

    I am trying to wrangle samtools into giving me a consensus, using reads mapped with bwa to a reference. After I generate a BAM, the results look pretty good using samtools tview:


    So, there is good coverage, etc.

    Now, when I try to generat a consensus, for example, by

    Code:
    samtools  pileup -6Aug sorted.bam |bcftools view -cg - | vcfutils.pl vcf2fq
    I get a whole lot of Ns in my consensus

    Code:
    @gi|159883886|emb|CU329670.1|
    nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
    nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
    nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
    nnnnnnnnnnnnnnnnnnnn
    +
    !!!!@@@@@@@@@@CEEEHHHHHHHHHHHHHHNKKNKQQQWWWWWWccux{{{{{~~~~~
    ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
    ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
    ~~~~~~~~~~~~~~~~~~~~
    I swear, I tried every combination of samtools and bcftools I could think of. Adding a reference sequence also doesn't make any difference. Any ideas would be most very much appreciated.

    Sasha

  • #2
    You didn't provide the reference sequences when calling pileup? If that's the case, all the ref bases are Ns and you probably wouldn't get the right consensus.

    Comment


    • #3
      Unfortunately, having a reference doesn't help:
      Code:
      samtools  mpileup -6Augf sequence.fasta -r "gi|159883886|emb|CU329670.1|:1-200"  sorted.bam |bcftools view -cg - | vcfutils.pl vcf2fq
      
      @gi|159883886|emb|CU329670.1|
      nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
      nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
      nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
      nnnnnnnnnnnnnnnnnnnn
      +
      !!!!@@@@@@@@@@CEEEHHHHHHHHHHHHHHNKKNKQQQWWWWWWccux{{{{{~~~~~
      ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
      ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
      ~~~~~~~~~~~~~~~~~~~~

      Comment


      • #4
        I'm not sure about your pileup issue, but see the thread "samtools tview reference sequence" regarding the string of N's in your tview. For me, this was caused by having a colon character in the reference sequence fasta header.

        Comment


        • #5
          I think I figured out what it was -- I had an old from a previous bam file hanging around. I re-indexed the file and everything was OK.

          Comment


          • #6
            Originally posted by Tally View Post
            For me, this was caused by having a colon character in the reference sequence fasta header.
            Wow, this sentence has solved a problem I have been having all morning.Thanks so much!

            Comment

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