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  • samtools help

    Hi,

    I had two Bam (1.bam, 2.bam) files from Bowtie2 and tried to call variants by Samtools. I have tried two procedures and they gave me different answers.

    Procedure #1:
    samtools mpileup -uf reference.fa bam1 bam2 | bcftools view -bvcg - > 12.bcf
    bcftools view 12.bcf | vcfutils.pl varFilter -D100 12.flt.vcf

    Procedure #2:
    samtools merge 12_merged.bam 1.bam 2.bam
    samtools mpileup -uf reference.fa 12_merged.bam | bcftools view -bvcg - > 12_merged.bcf
    bcftools view 12_merged.bcf | vcfutils.pl varFilter -D100 12_merged.vcf

    I can't figure out why. Please give some suggestions.

    Thanks very much. Happy Thanks giving day.

  • #2
    Well, in one case you're calling SNPs on multiple samples and in the other on a single sample with higher depth. I wouldn't expect them to give the same results.

    Comment


    • #3
      Thanks very much for your reply.

      I have one biological sample. And the Illumina sequencing will give me several FASTQ files, 1.fq, 2.fq... If I want to identify SNPs to reference genome, should I align them to genome separately, or combine the sequence first and align them as a single file?

      Thanks a million.

      Comment


      • #4
        It won't much matter if you align them separately and then merge or concatenate the files and then align the results (if you have paired-end reads, some aligners re-estimate the insert-size distribution throughout the alignment, so that could change things a bit). Whatever you do, don't create multiple BAM files from the same sample and then treat them as multiple biological samples.

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