variant calling using samtools -v- bcftools

ksc · 04-12-2011, 02:16 PM

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!

swbarnes2 · 04-12-2011, 02:23 PM

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

ksc · 04-13-2011, 07:44 AM

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.

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
Question on calling SNPs using samtools/bcftools	nkwuji	Bioinformatics	6	02-19-2013 09:52 AM
BFAST and Variant Calling	nexgengirl	Bioinformatics	8	01-02-2013 06:03 AM
minimum depth variant calling samtools/gatk	m_elena_bioinfo	Bioinformatics	1	12-06-2011 09:31 AM
variant calling	kjaja	Bioinformatics	1	11-04-2011 08:16 AM
Samtools variant calling questions	Chiel	Bioinformatics	2	06-07-2011 10:10 AM

04-12-2011, 02:16 PM	#1
ksc Junior Member Location: St. Louis, MO Join Date: Apr 2011 Posts: 4	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!

04-12-2011, 02:23 PM	#2
swbarnes2 Senior Member Location: San Diego Join Date: May 2008 Posts: 912	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

04-13-2011, 07:44 AM	#3
ksc Junior Member Location: St. Louis, MO Join Date: Apr 2011 Posts: 4	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.