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Old 04-12-2011, 02:16 PM   #1
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Location: St. Louis, MO

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Question variant calling using samtools -v- bcftools

I am new to samtools and have been trying out several protocols on the web for variant calling. Can someone please tell me what the main difference is between this step of the deprecated protocol:
samtools view -u aln.sorted.bam | samtools pileup -vcf Ref.fa - > aln.sorted.raw.txt

and this step of the new recommended protocol?:
samtools mpileup -ugf Ref.fa aln.sorted.bam | bcftools view -bcvg - > aln.sorted.raw.bcf

I thought that both of these steps should return the unfiltered set of putative variants; however, the first one gives me substantially more variants than the second. I just want to make sure I am solid about what exactly is happening at these steps so that I can make informed decisions about what filtering is necessary downstream.

Thanks for your help!
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Old 04-12-2011, 02:23 PM   #2
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Location: San Diego

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Mpileup by default adds a BAQ calculation, which pileup doesn't. Try the following, and see if the result is more like the pileup result

samtools mpileup -Bugf Ref.fa aln.sorted.bam | bcftools view -bcvg - > aln.sorted.rawB.bcf
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Old 04-13-2011, 07:44 AM   #3
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Thanks swbarnes2. That helped out a bit. However, I am still only getting half the variants using mpileup that I was using pileup. It is most likely that these are ones that would be filtered out in later steps anyway; however, it would be nice to know what is happening for sure.
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bcftools, samtools, variant analysis

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