SEQanswers

Go Back   SEQanswers > Bioinformatics > Bioinformatics



Similar Threads
Thread Thread Starter Forum Replies Last Post
Missing values in expression data matrix Amative Bioinformatics 1 03-18-2013 06:39 PM
How to use Samtools to output a list of SNPs (RS number) from a BAM file Michael Zhou Bioinformatics 3 11-20-2012 11:21 AM
VDB flag in VCF file lre1234 Bioinformatics 3 02-22-2012 05:32 AM
samtools/bcftools missing obvious SNPs? SamH Bioinformatics 3 01-17-2012 02:16 PM
missing reads in IGV display cswarth Bioinformatics 2 06-28-2011 08:30 AM

Reply
 
Thread Tools
Old 02-26-2013, 05:45 AM   #1
k2bhide
Junior Member
 
Location: West Lafayette

Join Date: Nov 2010
Posts: 5
Default Missing SNPs in samtools output eventhough it display VDB values

Samtools Version (0.1.18) error: I called SNPs for 17 samples from same organism, however some of the samples failed to list any SNPs on some of the chromosomes even though I expected some SNPs. Hence I looked in IGV browser and pileup information in order to verify whether there are any alternate alleles at the base position. It was clear that these positions had all mapped reads with alternate bases as compared to reference bases.

I removed v option from bcftools commands to get depth information for all the base positions irrespective of being called variant sites.

Here is the command I used
samtools mpileup -6 -Buf ../gff_and_Refgenome/ref.fasta in.bam | bcftools view -bcg -> in.var.raw.bcf
bcftools view in.var.raw.bcf > vcfutils.pl varFilter -D50 > in.var.fil.vcf
which gave following result for one of probable SNP positions, failed to be listed as SNP (AtoT) in final result file.

Chr2 617743 . A . 28.2 . DP=95;VDB=0.0535;;AC1=2;FQ=-30 PL 0

I would really appreciate if anyone would help me understand why such SNPs were ignored in final file eventhough it display VDB values which are related to SNP calling in samtools.
k2bhide is offline   Reply With Quote
Old 03-04-2013, 12:54 AM   #2
mbayer
Member
 
Location: Dundee, Scotland

Join Date: Mar 2009
Posts: 29
Default

Hi k2bhide,

here are a couple of things to try:

- try the mpileup without the -B option, in case this is filtering out SNPs
- try without the filtering script
- set the minimum base quality for mpileup to 0, in case the variants you are observing happen to be read errors
- set the SNP probability to low for bcftools (e.g. "-p 0.9999")

All of the above will make the command output a lot of dross/false positives but it is useful for the purpose of understanding what's going on.

You then need to tighten things up again to get your actual results. My approach is to make the initial SNP calling very relaxed and then filter appropriately, but if a SNP isn't there after the initial calling then it's obviously gone for good and you can't get it back.

I typically use something like this:

samtools mpileup -Q 0 -D -g -u -f <fastaFile> <bamFile> | bcftools view -vcgA -p 0.9999 - > SNPs.vcf

cheers

Micha
mbayer is offline   Reply With Quote
Reply

Tags
samtools vdb missingsnp

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 08:32 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO