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  • Question: bcftools: Wrong number of PL fields? nals=4 npl=6

    Hello all,

    I am new to bcftools and samtools, so forgive my question if it is ignorant. My goal is to extract consensus sequences between two different species.

    I was given paired-end Illumina sequencing data. So far, I used FastQC to trim and bowtie 2 to perform the mapping and used samtools to generate sorted bam files. We created two bam files by using single-ended bowtie 2 mapping - this was due to a large amounts of insertion-deletion polymorphisms between the two species.

    I tried the following to variant call:

    /home/sean/Desktop/assembly_software/samtools-1.3.1/samtools mpileup -d8000 -uf Mlongifolia_CMEN585_assembly_v1.0.fasta CMEN585.R_1.sorted.bam CMEN585.R_2.sorted.bam | bcftools call -c | vcfutils.pl vcf2fq > cnsnew.fq

    error: Wrong number of PL fields? nals=4 npl=6

    Then I tried using just a single bam file:

    /home/sean/Desktop/assembly_software/samtools-1.3.1/samtools mpileup -d8000 -uf Mlongifolia_CMEN585_assembly_v1.0.fasta CMEN585.R_1.sorted.bam | bcftools call -c | vcfutils.pl vcf2fq > cnsnew.fq

    error: Wrong number of PL fields? nals=4 npl=3

    What do these errors mean and how do I fix them? I have searched around, but have been unable to turn up anything so far.

    I assume you need to variant call before you extract consensus sequences. I tried to extract the consensus sequences anyway (just for kicks):

    /home/sean/Desktop/assembly_software/samtools-1.3.1/samtools \ mpileup \ -B \ -u \ -f Mlongifolia_CMEN585_assembly_v1.0.fasta\ CMEN585.R_1.sorted.bam | \ bcftools call -cg | \ vcfutils.pl vcf2fq \ ->R_1_consensus.fq

    error: Use of uninitialized value $l in numeric lt (<) at /usr/local/bin/vcfutils.pl line 566. Use of uninitialized value $l in numeric lt (<) at /usr/local/bin/vcfutils.pl line 566.

    Thank you for your help,

    Richard

  • #2
    The bcftools error is usually due to mixing different versions of samtools and bcftools. If you use samtools 1.3.1, then you need bcftools 1.3.1.

    I haven't a clue about the vcfutils error.

    Comment


    • #3
      Thank you for the reply. I tried to adjust the command so that it couldn't default to a different bcf tools version:

      /home/sean/Desktop/assembly_software/samtools-1.3.1/samtools mpileup -d8000 -uf Mlongifolia_CMEN585_assembly_v1.0.fasta CMEN585.R_1.sorted.bam CMEN585.R_2.sorted.bam | /home/sean/Desktop/assembly_software/bcftools-1.3.1/bcftools call -c | vcfutils.pl vcf2fq > cnsnew.fq

      Unfortunately, I got the same errors.

      Note: Neither --ploidy nor --ploidy-file given, assuming all sites are diploid
      [mpileup] 2 samples in 2 input files
      Wrong number of PL fields? nals=4 npl=6

      Richard

      Comment


      • #4
        I don't know if this helps, but I'm breaking down the steps currently to see what is failing.

        1. /home/sean/Desktop/assembly_software/samtools-1.3.1/samtools mpileup -d8000 -uf Mlongifolia_CMEN585_assembly_v1.0.fasta CMEN585.R_1.sorted.bam CMEN585.R_2.sorted.bam >file.mpileup

        2, /home/sean/Desktop/assembly_software/bcftools-1.3.1/bcftools call -c file.mpileup > file.vcf

        3, /home/sean/Desktop/assembly_software/bcftools-1.3.1/vcfutils.pl vcf2fq file.vcf > cnsnew.fq

        Currently, while running the first step I'm getting the message [mplp_func] Skipping because some_number is outside of 0 [ref:some_different_number. This might just be because the alignment is poor. I'm not sure.

        Thank you

        Comment


        • #5
          One last update:

          When I run mpileup, the command finishes. However, it only outputs this:

          ##contig=<ID=Contig12315,length=1495,IDX=12315>

          And then after a while starts to output this:


          1��!NA7<*>
          5�? ��?B �DA�B�A@D

          Sorry for the spammed posts, but I figure the more information the better.

          Richard

          Comment


          • #6
            One last update:

            When I run mpileup, the command finishes. However, it only outputs this:

            ##contig=<ID=Contig12315,length=1495,IDX=12315>

            And then after a while starts to output this:


            1��!NA7<*>
            5�? ��?B �DA�B�A@D

            Sorry for the spammed posts, but I figure the more information the better.

            Richard

            Comment

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