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Old 05-17-2011, 12:40 AM   #1
KNS
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Question 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
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Old 05-17-2011, 07:39 PM   #2
KNS
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can anyone help?
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Old 12-01-2011, 09:41 AM   #3
aslihan
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Did you find a solution to your problem? I have also similar questions.

Thanks
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Old 12-01-2011, 09:55 PM   #4
KNS
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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
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