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  • Issues with samtools mpileup and bcftools view

    Hello,

    I've been having some trouble interpreting the outputs from samtools variant calling pipeline. I'm working with a de novo transcriptome assembly and a .bam file containing the reads from 1 sample.

    When I run '$ samtools mpileup -g -f Trinity.adj.fasta NFS_6525_bowtie2.coordSorted.bam | bcftools view -bvcg - > NFS_6525_var.raw.bcf' I get a var.raw.bcf file that is ~275 million bytes of this output:

     ÿ BC —$…ßÎeÇQG¥ˆ+žÂ"—$æìÞÿF
    ‘,¡%‚+˱ÇÉ ÛÙˆái¹ ˧jÑY{õ¥å¯¦{wÿººzuuøå¯ÿâ'?ùßÿùÉOúõÛoÞ|ýîûo¿xÿÉ¿üò×ÿ¹|Üþò§?ýõ§ÿôÙ?þö“¿ûôWŸüó/~÷»ŸýêÍ_~ÿöOïß¾ûî“¿úÅ7ß|Ô*Þ¿ùþ‡þôÅ?¼ùê¯~öé¯þõ“Çßw˾øöý»wßüð/ýÿÆ_OÏOýÇ÷?|óö÷?ÿñ?þßýòÝ·ß~ñÝWŸÔôíŸzoþãOýüýüë>ûþíwoßÿ÷Ç_|

    I understand that a .bcf is in binary format but I am still unsure of the output.

    Then, when I ran $ nano on my .vcf file after running the command '$ bcftools view SFS_25428_var.raw.bcf | vcfutils.pl varFilter -D 100 - > SFS_25428_var.flt.vcf', I get a file that is 1.5 billion bytes of this:

    ^@^@^A^G^WT7<*>^@^Q^D^Q^Z^Q �^Q^P^@^@@A^@^@�@^@^@^@^@^@^@^@^@^@�mD^@^[iG^@^@^@^@^@^@^@^@^@^@�A^@^@�A^@^@^@^@^@^@^@^@^@^@�C^@��E^@^@^@^@^@^@^@^@^Q %^@^@�?^@^@^@^@^Q ^U^@^@�?^Q^K^U^@^@^@^@^Q^N1^@3^T�^$
    ^UZ�¾^Q^F^U^@^@�?^Q^G^U^@^@�?^Q ^U^@^@�?^Q^H^U^@^@�?^Q^K^U^@^@^@^@^Q^Na^@2^^<!^^�^@^@^@^F^@^@^@| ^A^@ݾ^A^@^@^A^@^@^@^@^@^@^

    During '$ bcftools view SFS_25428_var.raw.bcf | vcfutils.pl varFilter -D 100 - > SFS_25428_var.flt.vcf', my terminal window rapidly prints this to the screen:

    Argument "^A" isn't numeric in multiplication (*) at /usr/bin/vcfutils.pl line 302, <> line 1708539.
    Argument "^F" isn't numeric in addition (+) at /usr/bin/vcfutils.pl line 268, <> line 1708540.
    Argument "^F" isn't numeric in numeric ge (>=) at /usr/bin/vcfutils.pl line 268, <> line 1708540.
    Argument "^A" isn't numeric in multiplication (*) at /usr/bin/vcfutils.pl line 302, <> line 1708540.
    Argument "^F" isn't numeric in numeric ge (>=) at /usr/bin/vcfutils.pl line 268, <> line 1708541.
    Argument "^A" isn't numeric in multiplication (*) at /usr/bin/vcfutils.pl line 302, <> line 1708541.
    Argument "^F" isn't numeric in numeric ge (>=) at /usr/bin/vcfutils.pl line 268, <> line 1708542.
    Argument "^A" isn't numeric in multiplication (*) at /usr/bin/vcfutils.pl line 302, <> line 1708542.
    Argument "^F" isn't numeric in numeric ge (>=) at /usr/bin/vcfutils.pl line 268, <> line 1708543.


    Does anyone have any thoughts or input? I would really appreciate it. I'm a MSc level ecologist that was thrust into the fire of bioinformatics to forgive me if this is basic.

    Cheers,

    Mike

  • #2
    A vcf file should be plain text (unless compressed) so there is something wrong. What version of samtools/bcftools are you using? If not the latest I would suggest you try upgrading first.

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