Hi Dylan, check if the mpileup --gap-frac or --min-ireads options are perhaps causing it? Otherwise it is hard to tell without actually seeing the data.
Petr On Mon, 2017-04-03 at 16:24 +0000, Fox, Dylan wrote: > Hi, > > I am trying to call substitutions and indels in with samtools / > bcftools (versions 1.3.1 / 1.3.1-209-g1618245), and I am unable to > call an indel that is in the bam. Looking at the bam in a viewer > confirms that there is an indel at chr7 position 117559590 that > deletes a TCT sequence. Following the instructions on http://www.htsl > ib.org/workflow/#mapping_to_variant, I used a similar pipeline on the > indexed bam: > > samtools mpileup -x -d 1000000 -B -ugf <ref.fa> <sample.bam> | > bcftools call -vmO z -o <study.vcf.gz> > > I added flags parameters -x, -d, -B because they seem relevant to > this data, and adding them do not seem to cause this issue I’m > having. > Upon viewing the vcf, there does not appear to be any snp called at > chr7 position 117559590: > … > “ > chr7 117541810 . G T 11.4963 . > DP=1;SGB=- > 0.379885;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=0,0,1,0;MQ=40 GT:PL > 0/1:39,3,0 > chr7 117541812 . G T 10.5754 . > DP=1;SGB=- > 0.379885;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=0,0,1,0;MQ=40 GT:PL > 0/1:38,3,0 > chr7 117541813 . A G 11.4963 . > DP=1;SGB=- > 0.379885;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=0,0,1,0;MQ=40 GT:PL > 0/1:39,3,0 > chr7 117548628 . G T 210 . > DP=2032;VDB=0;SGB=- > 0.693147;RPB=0.925993;MQB=0;MQSB=0.9998;BQB=8.21384e- > 07;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=496,497,520,519;MQ=28 > GT:PL 0/1:243,0,255 > chr7 117559403 . A G 200 . > DP=344;VDB=0;SGB=- > 0.693147;RPB=0.995508;MQB=0.958341;BQB=0.736929;MQ0F=0;ICB=1;HOB=0.5; > AC=1;AN=2;DP4=175,0,169,0;MQ=41 GT:PL > 0/1:233,0,224 > chr7 117559479 . G A 222 . > DP=694;VDB=0;SGB=- > 0.693147;RPB=6.85819e06;MQB=0.95608;MQSB=1;BQB=0.986478;MQ0F=0;ICB=1; > HOB=0.5;AC=1;AN=2;DP4=180,179,167,168;MQ > =41 GT:PL 0/1:255,0,255 > “ > *** SHOULD BE HERE *** > “ > chr7 117587989 . T C 11.4963 . > DP=1;SGB=- > 0.379885;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=0,0,0,1;MQ=40 GT:PL > 0/1:39,3,0 > chr7 117587995 . T A 11.4963 . > DP=1;SGB=- > 0.379885;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=0,0,0,1;MQ=40 GT:PL > 0/1:39,3,0 > chr7 117587997 . A G 11.4963 . > DP=1;SGB=- > 0.379885;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=0,0,0,1;MQ=40 GT:PL > 0/1:39,3,0 > chr7 117587999 . G T 11.4963 . > DP=1;SGB=- > 0.379885;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=0,0,0,1;MQ=40 GT:PL > 0/1:39,3,0 > “ > … > I then used mpileup to generate a pileup file so I could confirm > whether mpileup was recognizing the snp. I used: > > samtools mpileup -x -d 1000000 -B -f <ref.fa> -o <test.pileup> > <sample.bam> > > This yielded this output at the region of interest: > “ > chr7 117559590 A 3686 .$..-3TCT..$.$.$.,-3tct,,- > 3tct,-3tct,,,,,,,,,-3tct,,,,,-3tct,-3tct,,-3tct,-3tct,-3tct,-3tct,- > 3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,- > 3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,- > 3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,- > 3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,- > 3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,- > 3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,- > 3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,- > 3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,- > 3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,- > 3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,- > 3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,,- > 3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,- > 3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,- > 3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,-3tct,- > 3tct,-3tct,- > 3tct,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, > ,,,,,,,,,,,,,,,,,,g,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, > ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,-3tct.-3TCT....-3TCT.-3TCT..-3TCT..- > 3TCT..-3TCT.-3TCT...-3TCT..-3TCT.-3TCT.....-3TCT......-3TCT.-3TCT.- > 3TCT..-3TCT..-3TCT..-3TCT..-3TCT.-3TCT.-3TCT.-3TCT..-3TCT..-3TCT....- > 3TCT....-3TCT.....-3TCT.-3TCT.....-3TCT...-3TCT..-3TCT.......-3TCT.- > 3TCT.-3TCT.-3TCT.-3TCT.-3TCT..-3TCT.-3TCT..-3TCT.-3TCT.-3TCT.- > 3TCT...-3TCT..-3TCT.....-3TCT.-3TCT.-3TCT.-3TCT.-3TCT...-3TCT.- > 3TCT.....-3TCT.-3TCT...-3TCT...-3TCT..-3TCT.-3TCT.-3TCT.-3TCT...- > 3TCT...-3TCT..-3TCT.-3TCT.-3TCT.-3TCT..-3TCT..-3TCT...-3TCT.......- > 3TCT...-3TCT.....-3TCT......-3TCT.-3TCT.....-3TCT.-3TCT..-3TCT.- > 3TCT...-3TCT..-3TCT..-3TCT......-3TCT.-3TCT.-3TCT.-3TCT.-3TCT...- > 3TCT..-3TCT.-3TCT..-3TCT.-3TCT..-3TCT.-3TCT.-3TCT.-3TCT...-3TCT..- > 3TCT.-3TCT..-3TCT..-3TCT.-3TCT.-3TCT.-3TCT..-3TCT. > ” > … > “ > HHHHH<HHH3HHHHHHHHFGHHH1HHFHH1HHHHHGHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHFHH > HHCHHHHHHHHHHHHHHHHHHHHHHFHHHHHFGCGHHHHHHHHHHHHHHHHFHHGHHHHFHHHHHHHHH > HHHHHHHHHHHHHHEHHH3HHHHAHHHHHHHHHHHHHHHHHHHHHHHHCHHHHHHHHHHHHHHHHHHHH > HFHHHHHHHHHHHHHHHHHFHHFHHDHHHHHHGHHHHHHHHHHHGHHHHHHDHHHHGGHHHHGHHH0HF > HHHDHHHGBHHHHHHHHGHHFHHGHHGHFHHHHFHGHHHHHHHHHFHHHHHHH3HHFHHGHHHEHFGHH > HHFHGH3HHHHHHHH2HHBHHHHHHHHHHHHFHHHHHHHGHHHH2HGFHHDHHHGHGDHHHFHGHH52H > HGHGHH2HHHFHGHGHGHHHFDHHHHHHHHHFFHHHHHHHHEHHHHHGHHHHHHHHHGHHHGFBHHHHH > HH2EH2HHHHHHHHHHGHHHHHFHGHHHHHHGHHHFHGHHHHHHFHHHHHHHHHHBHHH2HGFHFHHHE > HHHH2HHHHHHHHHHGGFHHHHHHHHHHHH2HFHHHHHHHHDHHHHHHHGHHHHHHHGH2HHDHHHHHH > HHFHHFHHHBHHFHHHHHHHEBHHHHHHHHHHHFHBHHHHHHHFHHHHHHHHHHHHHBHHDHHGHFHGH > HHGHHHHHHHFHH2FHHHHGHHHHGHHHHHHHHHHHHGBHHHHGGHHHHHHHHH2HHGHHHHGHHHHHH > HHHHFBHGHHHHHHHHHHHHHHFFHHHHHHEHHHHGBHHGFHHHHHHHHHHHHHHGHHHHHHHHHGHHH > HHHHHHHHGHHFHHHHHHGHFHHFFHHHGHHHHGHHAHFFHHHHHHHHH2HHHHHHHHHHDHHHHHGHH > GHHHHHHHHHHHHHHHHHHHHGHHHHHHHHHHHHHHGHHHHHHHHHHHHFHHHHHHHHHHHHHHHHHHH > FHHHHHHHHDHHHHHHHHHHHHHDHHHHHHHHHHHGHHHHGHHHHHHHHHHHHHDHHHHHHHFHHHHHH > FHHHHHHHHHHBHGHHHHHHHHHHHHHHHHHHHHHHHHHAH2HHHHHHHHGHHHHHHHHHHHHH5HHHH > HHHHHHH2EHHHHHHHHHHHHHHHHFHGGHHHHHHHGHHHHHHHHGHHHHHFHHHGHHHHHHHDHHHFH > HHHHHHHHFHHHFHHHGHHHHHHHHHHHHHHHHHHHHFFHHFHHHGHHHHHHHHHHGHHFHHHHHHBHH > HHHHHHHHHHHHHHHEHHHHHHHHHHHHHHHHHGHHHFHHHHHHHHHHHHHHHHHHHHHH2HGHH5HHD > FHHHGHHHHHHHHHFHHHHHHHHHHHHGHHHHHHHHHHHHHFHHHHEHHHHHHHHHHFHHHHHHHHHHF > HHHHHFHHHHHHHHHHHHHHHFHHH2HHHHHHFHHHHHHHHHHHGHHHHHHFHHHHHHDHHHHHHEHHD > HHHHH5HHHGHHHHHHHBHHHGHHHHHHHHGHHGHHGHHHFHHHHHHFHHHHGHDHDGHHHHHHHHHHH > HHHHGHHHFHHHHFHHHHHHHHHHHGHHHHHHHHHHHFHHHHHHHHHHHHHHHHHHHHHHHH2HHFF5H > HH2AHHHHHHHHGHGHGHHHHHHHHHHHHHGHHHHHHHHFHHHHHHHHHHGHHHHHGHHHHHGHHHHHH > HHHFHHHHHHHHHHGHHH5HHHHHHHHHFHHHHHHFFHHHHHD2DHHHHHHHGHHHHHHFHHHHGHHHH > HHHHHHHHHHHGHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHFDHHHGFHFHHHHHHHHHHHHHHHH > HFFHHHHHHAHHHHFHHHBHHHHHGHHHFHBHHHHHH5HDHBGHHHHHFHHHHHGHHHHHDHHGHHHHH > HHHHHHHHHHHHHHFHHGHFHDHHHHHHHHFHHGHHHHHHHHHHHH2HHHFHHHFA5GFGHHHHHHH2H > HHHHHHHHHHEHGGHHHHHH2HFBHHEHHEBHFHGHHHHHGHHHFHHHHHHGFHDBHGHHHHHHHDEH5 > HHHHHF2HHHFHHFHHHHHHHHHHHHHHGHH2FHHGHHHFGGHDHHHHHHHAHHHHFHHHHHHHHHHHG > HHHHHHGHHHHHH2FHHGHFHGHHHHHHFHHGHHHHGHHFHHHHHHDHHHHHHHHHHHHHHHHHHHGHH > HHFHHHHHHHHHHHHHBFHHFFHGHHHFHHHHHHHHGGHGHHHGHHHFHHFHHHHHHFHHHHHHHHHHH > 5HFHHHHHHGHHHGHHHHHHHHHHHHGHGFHHHHHFHHHHDHHHHHHHHHHHG2HHHGG5HHHHHHHHG > DHHHGHHHHHHHHHHHHHHHHHHHHDHHHEHHGBHHHHHHHHHHHHHHGHHHHHHHHHHHHHHBHHHHH > HHGHHFDHHFHHH5DHHGHHHHHGFHHFHHHHHHHHHHHHHHHHHFHHHHHHHFHHHHHH2HHHHHHHH > HHHHDHHFHHHHHAHHHHDHHHHFHHHHFHHHHHHHHHHGFHHHHHHFHFHHGH5HHHHHHHHHHHH2H > HHHHH2EHGHHHHHGHHHHHGHHHHHHHHHHFHHHHHHHHHHHHHHHHHHEHFHH2HHGHHHFHHGHHH > HHHGHHHH55HHGHHHHHGHHHFHHFHH2HHHHHHHHHEHFHGHHHHHHHHHHHHHHFHHHHHHGHHHH > HHFHGHHHHHHHHHBHHHHGHHHHHHHHHHHHHHHHHHHHBHHDHHHHHHHGGHHHFHHHHHHHHFHHH > GHHHHHHHHHHHHHFF2HHDHHHH5HGGHHHH2FHHHHHGHHHHHHGH5HHHHHHHHHGGHHHHHHHAH > HHHHHDHHHHHHHGHHGHHHGHHGHHHHHHHHHHHGHHHHHHHHHFHHHHHFHGHDHHHHHFHHHHHGH > GHGHDHFHHHHHHHHHHHHGHHHHHFHHFHHHH2HHHHHFH5HHHFHHFHHHHHHFHHHAHGGHHHHHH > BHHHHHHHHHHHFHHHGHGHHHHHHHHHHHHHHHHHHHHHDHHHHHHGHHHHHFHGHHHHHFHHHHHHH > HHHHHHGHHHFHHHFHHHGFHHFHHHHHBHHHGEHHHHHHHBHHHHHHHFHHHHFHGBHHHHFHHHHHF > HHHHHHHHHHHHHHHHHHHHHFHHHHHHHHHHHHHHHHDHHHGHHHHHHHHHHHHHHHHHHHHHHHHHH > GHHHHHHHHHHHGHHHGHHHHFFHHHHHHHHHHHBHHHHHHHHHHGGHHHHHHHGHDHHHHHFHHHHHH > HHHHFFHHHGHHHHHFHHHGAHHHHHHGHHHHHHH5HHHHHHHHHHHHHHHHHEFHHBHHHHFHHHHGH > HHHHHHHGHHHFHHFHHHHHHFHHFHHHGHHHFHHHEHHHHHHHHHHDHHHHHHHGHHHFFHGHHHFHH > HGHHHHHHHHGHHHHFHHHHFHHHHHFHHHHHHHFHHH2GHFHHHHHHHHHHGHHHFHHHHHHHHHHHH > HGHBHAHHHHHHGHHHAHHHHHGHHFFHHHHHHHHHHHHHGHHHHHFHHHHGHHGHHHDHHFHHHHHHH > HHHHHHHHEHHHHHHHHAHHHHHHHHHHHHHHBHHHHHHHHFGHFHHHHHHHHHFH2GHHHHHHGHHFH > HHHHGHHHHHHHHHHHGHDHHHHHHHHHHHHHHHHHFHHHHHH2HHHFDHHGHHHHHGHGDHHHHHHHH > HHHHHHFHHHHHHHHHFHHHHHHHHHHGHFHHHHHHHHHHHHH5HH2HEHHHHGBHFGGGGHHBHBFHG > HBHHHHAGHHHHHHDFG2HHHHHHHHHGH > “ > > This pileup data is consistent with the bam viewer I used, yet > somehow there is a disconnect in calling the indel in the final vcf. > Hopefully I’m just missing a flag or parameter. Any thoughts on why I > am not able to call this snp? Any help will be greatly appreciated! > Thanks, > -dylan > > ------------------------------------------------------------------- > ----------- > Check out the vibrant tech community on one of the world's most > engaging tech sites, Slashdot.org! http://sdm.link/slashdot > _______________________________________________ > Samtools-help mailing list > Samtools-help@lists.sourceforge.net > https://lists.sourceforge.net/lists/listinfo/samtools-help -- The Wellcome Trust Sanger Institute is operated by Genome Research Limited, a charity registered in England with number 1021457 and a company registered in England with number 2742969, whose registered office is 215 Euston Road, London, NW1 2BE. ------------------------------------------------------------------------------ Check out the vibrant tech community on one of the world's most engaging tech sites, Slashdot.org! http://sdm.link/slashdot _______________________________________________ Samtools-help mailing list Samtools-help@lists.sourceforge.net https://lists.sourceforge.net/lists/listinfo/samtools-help
