For some reasons, an empty line was added at the bottom of the index genome file. Remove it solved the problem…
Antoine > On 09 May 2016, at 03:08, Petr Danecek <p...@sanger.ac.uk> wrote: > > Different reference sequence might have been used for alignment and > calling. You can check with `samtools view -H` whether the @SQ lines are > as expected and with `samtools tview` whether the alignments look > reasonable. > > Petr > > On Thu, 2016-05-05 at 11:31 -0400, Antoine Paccard wrote: >> Thanks Petr. I actually did not see that. What do you think could cause >> this? I am using the latest release of my reference genome. sam files >> from my BWA alignment looks normal and contain position of reads on the >> genome (scaffold…etc). Maybe that version of the reference genome is >> causing an issue? It’s hard to tell. > >>> On 05 May 2016, at 11:01, Petr Danecek <p...@sanger.ac.uk> wrote: >>> >>> There seems to be a problem with your reference sequence. The VCF >>> fragments you sent have all REF=N at all positions. These are not called >>> unless --keep-masked-ref is given to bcftools call. >>> >>> On Thu, 2016-05-05 at 10:34 -0400, Antoine Paccard wrote: >>>> Thanks Petr. I have attached heads and tails of both BCF and VCF files >>>> (created with samtools view). Please let me know if you see something >>>> strange. Also, I have a couple of bam files that are much bigger >>>> compared to the other ones (2GB against 250MB). I’m not sure if this >>>> could cause the issue. >>> >>>> Thanks for your help! >>>> >>>> >>>>> On 05 May 2016, at 10:23, Petr Danecek <p...@sanger.ac.uk> wrote: >>>>> >>>>> Can you make part of your raw mpileup output available for me to take a >>>>> look at? >>>>> >>>>> Petr >>>>> >>>>> On Thu, 2016-05-05 at 10:06 -0400, Antoine Paccard wrote: >>>>>> Hi Petr, >>>>>> >>>>>> Thanks for your answer. I tried your suggestions but obtain the same >>>>>> result: an empty VCF file containing only the header. I suspect that >>>>>> something is going wrong with mpileup or with my version of bcftools. >>>>>> Creating a VCF out of my BCF using samtools view, I confirm that my VCF >>>>>> just looks completely normal. I also confirm that I am using versions >>>>>> 1.3.1 and that samtools, bcftools and htslib are all in my PATH. >>>>> >>>>>> Please let me know if you have any other suggestions. >>>>>> >>>>>> Antoine >>>>>> >>>>>>> On 05 May 2016, at 09:47, Petr Danecek <p...@sanger.ac.uk> wrote: >>>>>>> >>>>>>> Hi Antoine, >>>>>>> >>>>>>> the usage seems alright. An empty output can be produced when there are >>>>>>> no high confidence calls, try to run without the -v option to get also >>>>>>> non-variant sites on the output. >>>>>>> >>>>>>> Then you can also try "bcftools call -mv". If there is still no output, >>>>>>> try with bigger value of -P, e.g. "bcftools call -mv -P0.01". >>>>>>> >>>>>>> Petr >>>>>>> >>>>>>> On Wed, 2016-05-04 at 12:22 -0400, Antoine Paccard wrote: >>>>>>>> Hello, >>>>>>>> >>>>>>>> I am trying to produce a vcf file using bcftools call but it produces >>>>>>>> an empty vcf file containing only the header. In short, here is what I >>>>>>>> do: >>>>>>>> >>>>>>>> 1. Alignment with BWA >>>>>>>> 2. With samtools, make sorted.bam files >>>>>>>> 3. With samtools, index the sorted.bam files >>>>>>>> 4. Run samtools mpileup in the following way: >>>>>>>> samtools mpileup -C 50 -E -t SP -t DP -u -I –f /genome/refgenome.fa -b >>>>>>>> bam_list.txt > output.bcf >>>>>>>> 5. Run bcftools call: >>>>>>>> bcftools call -v -c output.bcf > output.vcf >>>>>>>> >>>>>>>> I am using versions 1.3.1 of samtools, bcftools and htslib. I tried >>>>>>>> reinstalling these programs but it did not change the issue. I also >>>>>>>> tried with versions 1.2. Same problem. As far as I know, the bcf file >>>>>>>> seems fine, it contains lots of data and is 20GB. >>>>>>>> >>>>>>>> I tried producing a basic vcf file using bcftools view: bcftools view >>>>>>>> output.cf > output.vcf and it works. The vcf file seems completely >>>>>>>> normal. >>>>>>>> >>>>>>>> Could anyone help me with this? Why would bcftools call produce an >>>>>>>> empty output? >>>>>>>> >>>>>>>> Thanks >>>>>>>> ------------------------------------------------------------------------------ >>>>>>>> Find and fix application performance issues faster with Applications >>>>>>>> Manager >>>>>>>> Applications Manager provides deep performance insights into multiple >>>>>>>> tiers of >>>>>>>> your business applications. It resolves application problems quickly >>>>>>>> and >>>>>>>> reduces your MTTR. Get your free trial! >>>>>>>> https://ad.doubleclick.net/ddm/clk/302982198;130105516;z >>>>>>>> _______________________________________________ >>>>>>>> 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. >>>>>> >>>>> >>>>> >>>>> >>>>> >>>>> -- >>>>> 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. >>>> >>> >>> >>> >>> >>> -- >>> 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. >> > > > > > -- > 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. ------------------------------------------------------------------------------ Mobile security can be enabling, not merely restricting. Employees who bring their own devices (BYOD) to work are irked by the imposition of MDM restrictions. Mobile Device Manager Plus allows you to control only the apps on BYO-devices by containerizing them, leaving personal data untouched! https://ad.doubleclick.net/ddm/clk/304595813;131938128;j _______________________________________________ Samtools-help mailing list Samtools-help@lists.sourceforge.net https://lists.sourceforge.net/lists/listinfo/samtools-help
