SEQanswers

Go Back   SEQanswers > Bioinformatics > Bioinformatics



Similar Threads
Thread Thread Starter Forum Replies Last Post
SAMtools missing bcftools khadeeja Bioinformatics 8 06-19-2013 02:06 AM
Question on calling SNPs using samtools/bcftools nkwuji Bioinformatics 6 02-19-2013 08:52 AM
Samtools mpileup/bcftools cristae8 Bioinformatics 3 05-02-2012 12:43 PM
Problem with samtools/bcftools alma Bioinformatics 4 01-06-2012 12:40 AM
samtools/bcftools problem? nimmi Bioinformatics 0 01-12-2011 11:59 AM

Reply
 
Thread Tools
Old 01-11-2012, 04:56 PM   #1
SamH
Member
 
Location: Moscow, ID

Join Date: Sep 2010
Posts: 16
Post samtools/bcftools missing obvious SNPs?

Hi, I am having some trouble with what appears to be missed calls for SNPs while using the samtools/bcftools package.
I am using the latest release samtools 0.1.18 (r982:295), and bcftools 0.1.17-dev (r973:277). My reads are SE Illumina, which have been trimmed of low-quality ends and discarded if they contain low-quality internal bases. Reads are ~55bp long, and coverage is 300-500x per sample.

I first aligned against a reference genome using bwa and sorted using samtools:
bwa aln -t 3 ./ST3-index/NC_015433_mod.fna ../03-Trimmed/s_3_trimmed.fastq > s_3.aln.sai
bwa samse ./ST3-index/NC_015433_mod.fna s_3.aln.sai ../03-Trimmed/s_3_trimmed.fastq | gzip > s_3.sam.gz
samtools view -uS s_3.sam.gz | samtools sort - s_3

I then calculated coverage:
samtools mpileup -BQ0 -d10000000 -f ./ST3-index/NC_015433_mod.fna s_1.bam s_2.bam s_3.bam > SSIM_1-2-3_coverage.mpileup

As well as unfiltered variants:
samtools mpileup -uf ./ST3-index/NC_015433_mod.fna s_1.bam s_2.bam s_3.bam | bcftools view -bvcg - > SSIM_1-2-3_variants.bcf
bcftools view SSIM_1-2-3_variants.bcf > SSIM_1-2-3_variants.vcf

I then started spot checking some of the more interesting variants using a bam viewer (tablet) when I noticed something odd. One of the variants which showed up in the bam file, wasn't listed at all in the vcf file. I checked this position in the mpileup file, and it appears as if it should be an obvious SNP with very deep coverage. I am hoping that someone can please help me understand why this SNP wouldn't be called by bcftools.

Relevant mpileup line for the missing SNP (position 569984 in ref) is:
NC_015433.1_Ss_ST3 569984 A 376 C$C$C$c$c$CCcCCCccCcccCccccCCcccccCccCCcccCCCCCccccccccCcCCCcccCCCCCCCcCCcCCccccCCCccccCccCCCccCCCcCcCCCcCCccCCCCCccccccccCCccccCccccCccccccCCccccCCcCCCCcccccCCCcCCCCCcCCCCCcccCCCcCcCCcCcCcCcCcccCCCccCCccCCcCcCCCCCcCCCCCcccCCccccccccCCCCcCCCcccCCCcccCCcCCCCccCccCCCCCCCccccCCCccCCCcccCCCCcCCCcccCCCCCCcccCCCCccCcCcCCCCCCCCCcCccCCCCCCCcccccCCCCCCCccCCcCCCccCccCCCCCccccCCcCcccc^FC^FC^Fc^FC^Fc hhhghgghgghfhhfhggehdhhhhhfhhhffgfchYhhghhfhhhghghcfgghhhhcghhhggchgfchhhffhggh_ffhhggghffcghghfafhhhhchhhhhhchgghcgghfhhffffa_chdhhghhhhhfhhfgfghhgghhhghhfghfhghhchgfghhhhhghhfhhfghhhgdffgghhghfhhchhhhfgfhgggghfgghhfhghfhfhdhhhhg^ghchhfeghhhhhhhghgggghghhhhgdghhhhhfhhhghhhchhgcgghhgghhcghehhfh]hhgfhhhhhhhhhghhgghgafehcgfhhhhghhhfhhhhhhdghhhhhhhfhhhhfhhhhhhcghgh[ghhgh_hh`hh 505 C$C$C$C$C$C$c$c$C$C$CCcccccctccccCCCCCcccCccccCCcccccCCCcCcccCCCCcccccCCCcccccCCcCccCCCcCccccCCcCcCccCCCCcccccCcCcCccccccccccccccccCCCCCCcCcccCCCccccccCCccCcCCCCCCcccCCCCCCCCcccCCCCCCccCcCccccCcccCccCCCCcccCCCCcccCCCCCcCCcccCCCCCCCcccccCCCCCCCcCccCcccccCCCCCcccCCCCCCCCcCcCcCcccCCCCCCcCcCCCCCCcCcCcCCCccccCCCCCCccCCcCCCCCccCCCCCcCCCCCCccCCCccCCCCCCCccccccCccCCcccCCccccCcccccCccCcccCcCCCcccCCCCCCCcccCcCCccccccCCcccccCccCCCCCccCccccCCCCcccccCCcCCCcCCCCCCCccccccCcCCCcccCcCcccCCCCCCCccccCCCccCccCCCCCcccCc^FC^FC^FC^FC^Fc^Fc^Fc^Fc^Fc^FC^Fc fhhhggghhgghdhhfhhhhhghhcchfhhhfhhhhhhhhhh_hghhhhhhhhhfgaffhhhheghdfdghdhhgggfhfhhhgefhdhf_ghghfh_ghghghcfhhghchhghgggfgchghghhhhfYdfghfagggehafggfdhhhgde_hghhhhhhhhfefhfhhhhhfggahhdhhhhgffhhhhfhfhhhhfhhhhghfahhgghfhhhhhhgehhhedhhfhdehghafghhhhhghhhfhhhhhdhehghghhhhhhgghhhghhhhhfhhhhhhhhhhhhghhhhhhhhhhhhhfhfghhhggghhhhfhghggehaghhgh_hhhhhhchgfhhchhfhhdfhfhhhhhhhbhhghhahhghhhhaghghhhhhhh\hhfhhhhghhhhghhfhhhghhfh_cchhhhghghhffhhhahhgcgehhfhghhhdghhhhhhhfhhehhhhghhchhfhchfhfhhhhhhhfchhhfggdhghgfhhhghhhf 447 C$C$C$C$C$C$c$CCCCCCcccCCCccCCCCCCccccCcCccccCcCCccccCcccCCCCcccCCcCCCCCcCcccCCcccCCCCCCCCCCCcCccccCccCCcccCcCCcCccccCCcccCcCcccCCCccCccCCccccCccCcCcccCCccCCcccccCCCCCcCccCCccccCCCCCcccccCCCcCcCCCCCccCcCCCcCCCccccCCCCCcCcCccCCCCCCccCCCCCCccCcccccCCCCCCCCCCccccCCcCCCCCCCccCCCCCCCCcccCcCCCCCCcCCCCCccccccCccCCCCcccccCCcCcCCCCccCCccCCCccCCccccCCCCCCccCCCCcCCCCCCCCCCcCCccCccCccCCCccccccCcccCCCCCCCCccccCCCCcccCcCcccCCCCCCCCcccccCCCCCCccCcCCCCcc^FC^FC^FC^FC^FC^FC^FC^FC^Fc^FC^Fc^Fc hfhhghhhhfhhfhhhfhghhhhhchegfhaghhhhhhhhhgghfgcfhhhhghghhhhghhgfchfhghhgghdhgfhhhhghggghhhhfhg\gfhcehhghfh^Whfdhggghfgchhhhfhahgfchgfgghgghghhgeehdhdggdfffhhfhghggfgdfghhfhghhhhehfghhhhhehghhhdhhghdgghggdchhhghhhhhhhhhhhhhggghgdhhfghfgahfhghfhhhehhhghfhfhhhhhhcghhhghgghhhhahhhffgdghhghhgghhghghhchhhhhhhhfhgcfhh^fhghhhhhhfhhghfhhfhchhgghghhhhhhffahhg^hgghhhhhfghhggdch\hhhhhcffhghhghhhhhgfhgghdghhhhehhhhhhhhhgffahghhhhfahchgghgfhhghhhfhhhchfghgd

A nearby SNP which did show up in the vcf file and is very similar:
NC_015433.1_Ss_ST3 542551 A 237 g$GGGgGGgGgGGGggggGGGGGGGggGGGGGggGgGgGGGGGGgggGgggGGGGGGgggGggGGGGGGGggGgGGgGgGGGGGGgggGGGGggGGGGgGGGggGGggGggGGggggGggggGggggGGGGGgggGgGGGgggGgGGGGGggGGGgggGGGGgggGgGgGggGggggGgGgggGGGGGGggGGGggGgGGGGgGGGGGgGGgGgGGGGgGGGGGgGGGGGGGggGggg hhfhhfghhhfdghhghcfgfffbhhfgchhhhghhhhfhhhahgfdhhdfhfeffgggfhghhhhghhfhfhhhhfhggf]gfhghhhhhgh_fdhhfgghaggagghhfghhhhdhghgghhhghgggchhhggggghhgddhgafgahhhgfhgfggfghhfhfhfhhchghhg\fhhhcffgcgghfdchgchggfghhfhdhhgghfhhfhghfhhffhfhghgfghhdhhh 270 G$G$G$g$g$GGGGGGgGgGGGGGGggGGGgggGGgggGGGgGgGgGGGGGGgGGgGGggGGGGGGggggGGggGGGGggGGGGGGgGgGGggggggGGgGGGGGGgggGGgGggGgGggggGggGgGGGGggGgggGGGggggGGGGGGgGGGGGGGGGgGggggGGGGGgGGGGGggGggGGGGGGgggGgGGGGgGgGGGGGGGggGggggGGGGGGGGgggGgGGGGgGGGGgggggGGGGGGGggGGGGGGGGGGGGGGGGGGGGggGGg dhhhgfgehffhghdffccghh\chhhgfchghfhahhhhgchehgdhcghhfghgeffdhhfhhhgghhhhfhhcfhdfhhfffcchghhhfghhhfhffhehdchhcgdhhhhhgghhcgfhfhhhcghhhffhhhhfgggghhghdeggggchchhhhhfggf\egfcgghghhhhffffhhhfh\dfdhfhfgffgfahg\hhhhdfcc_affhfhgfhffhhfhhhhhhhgdffhdffhhghhhghhghhfafghchfhhfgfXh 285 G$G$G$g$g$GGggGGGgggggggGgGggggGggggGGgggggggggGGGggGGgGgGGggGGGggGGGGGGggGGgggGGGGGGggGggGGGGGGGGgGggGgGGGgGGGGgGgGgGGGgggggggGggggGGGGGGgggGggGGGggGGGGGGgggggggggGggGggGGGGGGGGGGggggGGGGggGgGggGGgGGggGggggGGGGGggGGGGGGGGGGggGGGGGgggGGGGGGgggGGGGggggGGGGggggGgGgggGGGGgGGGGGGGGGgGGGGGGGGgg gchhffhgh^gfhghhhhgdecghhfdhhhhdfhhffhhhhgbfhhfehhfhc^hhfffhhcfdhchhhaahfhggghfdghfhegfhfdefffehhghghYhdgahhfhhhfffhhhhdhhfhhchcgffffhhggfhhhdgcdfgfgfhhghahghhghcahdgghhfffgdfhhhhefgfhhghchhhfgfchhghhggfgdffhhfdffffffafhhgfffchhhfafddfhfhcgcfhhhfffhfchggfgfhhhhhfhhhhhhbhhhhhhhhf\fhdfh


And the two lines from the vcf file which should be flanking the missing SNP:

NC_015433.1_Ss_ST3 543182 0 T G 999 0 DP=1575;VDB=0.0742;AF1=1;AC1=6;DP4=1,0,597,717;MQ=37;FQ=-260;PV4=0.45,7.8e-131,0.46,0.4 GT:PL:GQ 1/1:244,255,0:99 1/1:235,255,0:99 1/1:217,255,0:99 2 2 2
NC_015433.1_Ss_ST3 570005 0 C T 999 0 DP=1403;VDB=0.0710;AF1=0.6667;AC1=4;DP4=239,177,498,334;MQ=37;FQ=999;PV4=0.43,0,0.24,1 GT:PL:GQ 0/0:0,255,255:99 1/1:255,255,0:99 1/1:255,255,0:99 0 2 2

Although I have only discovered one of these cases, I am concerned that I might be missing other variants as well.

Thanks for taking a look.

Sam
SamH is offline   Reply With Quote
Old 01-12-2012, 03:01 AM   #2
colindaven
Senior Member
 
Location: Germany

Join Date: Oct 2008
Posts: 415
Default

Try running Samtools mpileup with the BAQ option turned off (-B) and checking the same position. The SNP may have been masked.
colindaven is offline   Reply With Quote
Old 01-17-2012, 06:16 AM   #3
rand
Junior Member
 
Location: Sweden, Stockholm

Join Date: Jul 2009
Posts: 4
Default

Thanks, it helped me.
rand is offline   Reply With Quote
Old 01-17-2012, 02:16 PM   #4
SamH
Member
 
Location: Moscow, ID

Join Date: Sep 2010
Posts: 16
Default

Yeah, adding -B made that SNP appear:

NC_015433.1_Ss_ST3 569984 . A C 999 . DP=1328;VDB=0.0735;AF1=1;AC1=6;DP4=0,0,657,577;MQ=37;FQ=-288 GT:PL:GQ 1/1:255,255,0:99 1/1:255,255,0:99 1/1:255,255,0:99


thanks for the tip!

Sam
SamH is offline   Reply With Quote
Reply

Tags
bcftools, mpileup, samtools, variant

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 08:58 AM.


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