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  • differences in coverage BEDtools - samtools mpileup

    Hi,
    I have been using samtools mpileup and BEDtools coverageBed under default settings to count coverages at SNP sites identified with samtools mpileup. Both programs used the same paired BAM files produced by the Bioscope reference mapper as input. Samtools mpileup should report coverages under the DP and (quality checked) DP4 values in the vcf-fileoutput, while coverageBed reports all features in the file covering identified sites. I noted that BEDtools counts are much higher than those reported by samtools.

    What am I getting wrong? Or if not, based on what criteria are reads counted? Any suggestions much appreciated!

    samtools 0.1.16 (r963:234):

    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

    coverageBed (v2.12.0):
    samtools view -b /path/to/folder/A_paired_sorted.bam | ./coverageBed -abam /path/to/folder/A_paired_sorted.bam -b /path/to/folder/SNPs_zerobased.bed > /path/to/folder/counts_A_s.txt

    fileformat SNPs_zerobased.bed file(includes only sites which interest me):
    scaffold_1 7354 7355

    example vcf line (4 separate samples sequenced):
    scaffold_1 7355 . A T 257 . DP=15;AF1=0.8585;CI95=0.625,0.875;DP4=0,1,0,12;MQ=34;FQ=-7.6;PV4=1,0.34,0.058,0.41 GT:PLP:SP:GQ 1/1:36,6,0:2:0:11 1/1:82,9,0:3:32668:13 0/1:81,0,28:4:8249856:24 1/1:101,12,0:4:0:16

    separate counts in coverageBeds for each sample
    scaffold_1 7354 7355 4 1 1 1.0000000
    scaffold_1 7354 7355 5 1 1 1.0000000
    scaffold_1 7354 7355 6 1 1 1.0000000
    scaffold_1 7354 7355 6 1 1 1.0000000

    counts from samtools mpileup: DP=15 (DP4=13)
    counts from coverageBed: 21

  • #2
    can anyone help?

    Comment


    • #3
      Did you find a solution to your problem? I have also similar questions.

      Thanks

      Comment


      • #4
        Alishan,
        two ways to get at counts etc.
        1) Use one of the old samtools versions - 0.1.12a seemed ok.
        2) Even better: Use GATK

        Comment

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