A heretically simple approach to variant calling

krawitz · 12-21-2011, 12:16 AM

Hi everyone,

we had a look at the distribution of heterozygous allele frequencies in NGS datasets and found that their variance is larger than expected by a bionomial distribution (http://www.ncbi.nlm.nih.gov/pubmed?term=22127862). For every variant caller this means a binomial prior distribution is not the right choice and might lead to false negative calls. We also found that a simple frequency classifier (heterozygous if covered by more the 20 reads and variant allele between 14% and 86%) is more sensitive at comparable specificity for high quality data, compared to default setting of most standard calling tools.

Is anyone aware of a fast tool, that allows to apply such a frequency filter directly on a .bam file?

cheers,

peter

Hena · 12-21-2011, 03:58 AM

You can ask samtools mpileup to print out the nucleotide pileups for each position in bam file. Parsing that should be fairly simple with a script.

krawitz · 02-04-2012, 05:07 AM

the samtools mpileup output can be piped into VarScan to apply a coverage and frequency filter:
samtools pileup -f reference.fasta myData.bam | java -jar VarScan.v2.2.jar pileup2snp --min-coverage 20 --min-var-freq 0.14
see: http://varscan.sourceforge.net/using-varscan.html

NGSfan · 04-24-2012, 09:10 AM

Very nice paper.

Sometimes simpler is better.

Rocketknight · 04-25-2012, 04:01 AM

This is awesome. Moving away from big fancy well-established tools to something like the "14-86%" rule is scary though.

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12-21-2011, 12:16 AM	#1
krawitz Member Location: Bonn Join Date: Feb 2010 Posts: 30	A heretically simple approach to variant calling Hi everyone, we had a look at the distribution of heterozygous allele frequencies in NGS datasets and found that their variance is larger than expected by a bionomial distribution (http://www.ncbi.nlm.nih.gov/pubmed?term=22127862). For every variant caller this means a binomial prior distribution is not the right choice and might lead to false negative calls. We also found that a simple frequency classifier (heterozygous if covered by more the 20 reads and variant allele between 14% and 86%) is more sensitive at comparable specificity for high quality data, compared to default setting of most standard calling tools. Is anyone aware of a fast tool, that allows to apply such a frequency filter directly on a .bam file? cheers, peter

12-21-2011, 03:58 AM	#2
Hena Member Location: Finland Join Date: Nov 2009 Posts: 19	You can ask samtools mpileup to print out the nucleotide pileups for each position in bam file. Parsing that should be fairly simple with a script.

02-04-2012, 05:07 AM	#3
krawitz Member Location: Bonn Join Date: Feb 2010 Posts: 30	the samtools mpileup output can be piped into VarScan to apply a coverage and frequency filter: samtools pileup -f reference.fasta myData.bam \| java -jar VarScan.v2.2.jar pileup2snp --min-coverage 20 --min-var-freq 0.14 see: http://varscan.sourceforge.net/using-varscan.html

04-24-2012, 09:10 AM	#4
NGSfan Senior Member Location: Austria Join Date: Apr 2009 Posts: 181	Very nice paper. Sometimes simpler is better.

04-25-2012, 04:01 AM	#5
Rocketknight Member Location: Ireland Join Date: Sep 2011 Posts: 86	This is awesome. Moving away from big fancy well-established tools to something like the "14-86%" rule is scary though.