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  • vcf to consensus call reference instead of N

    Hi all

    I have a few genomes sequenced using illumina. I have used samtools and vcfutils to make a consensus for each. All pretty standard stuff. However using vctutils to make the consensus gives me far to many N nucleotides to be happy with.

    Is there a way that i can create this consensus but where it currently has provided an N nucleotide it actually inserts the reference nucleotide in its place?

    Your help would be much appreciated. Any ideas?

  • #2
    Have you tried samtools mpileup?

    Comment


    • #3
      Sorry thats what ive used in the process. I pretty much used this exact command:

      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

      Anything i can change there to get it to call the ref seq rather than N's?

      Comment


      • #4
        Normally you get those Ns when you don't specify the reference or specify a different version of the reference file in the command line.

        One thing I would check is if the mpileup is producing the reference bases at each position and not Ns without piping the first output to bcftools.

        Comment


        • #5
          Thanks for the response i'll test the mpileup tonight.

          I'm pretty sure i have the reference sorted. I've used snpEff to analyse the SNPs in the genomes and for each SNP it has had the correct reference sequence.

          Could be be a problem of coverage? as in the sequencing reads might not cover this area?

          Comment


          • #6
            Could be coverage, but if that's the case then it shouldn't be making a variant call at that site right with a certain degree of confidence right?

            Comment

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