The clue may be in how you did "The fastq files were filtered to remove bases with score less than 20"
On 5 November 2014 04:39, Arumilli, Meharji <mehar....@gmail.com> wrote: > Dear Ryan, > The fastq files were filtered to remove bases with score less than 20 > and then aligned using bwa mem. Followed by indel realignment, fix-mate > pair, BQSR using GATK and then variant calling. > On 04/11/14 22:30, Devon Ryan wrote: > > Having the occasional N in a read is pretty common. The bigger mystery > is how it got a Phred score of F even with BAQ disabled. It would seem > unlikely for the sequencer to have actually assigned an N that score. Were > these fastq files somehow massaged prior to alignment? > > > > Devon > > > > ____________________________________________ > > Devon Ryan, Ph.D. > > Email: dpr...@dpryan.com > > Tel: +49 (0)178 298-6067 > > Molecular and Cellular Cognition Lab > > German Centre for Neurodegenerative Diseases (DZNE) > > Ludwig-Erhard-Allee 2 > > 53175 Bonn, Germany > > > > On Nov 4, 2014, at 9:15 PM, Arumilli, Meharji wrote: > > > >> Hi, > >> > >> I have attached two screenshots from samtools tview. One of them is > normal where the variant is not called with reference sequence on the top > and other with variant called as "AN". Does this help to infer further? > >> > >> On 04/11/14 21:26, Thomas W. Blackwell wrote: > >>> So what could possibly have introduced that 'N' into the sequence > reads ? Is it present in the original .fastq file ? > >>> > >>> - tom blackwell - > >>> > >>> On Tue, 4 Nov 2014, Arumilli, Meharji wrote: > >>> > >>>> Hi, > >>>> > >>>> These are the commands used to call variants using samtools-0.1.19: > >>>> > >>>> samtools mpileup -ABugf ref.fa -l bed -d 1000000 bam | bcftools view > -vcg - | vcfutils.pl varFilter -D 1000000 > out.vcf > >>>> vcfutils.pl varFilter -Q 40 -d 10 out.vcf | awk '$6>=40' > fin.vcf > >>>> > >>>> > >>>> Hope this might help to some extent. > >>>> > >>>> > >>>> On 04/11/14 20:52, Thomas W. Blackwell wrote: > >>>>> As with the earlier question, we are all puzzling what could > possibly have introduced N's into the sequence reads. No details of > upstream processing steps are given, so no one has any ideas to contribute. > Simplified command lines and software version numbers are always helpful. > >>>>> > >>>>> - tom blackwell - > >>>>> > >>>>> On Tue, 4 Nov 2014, Arumilli, Meharji wrote: > >>>>> > >>>>>> Hi, > >>>>>> > >>>>>> > >>>>>> I have performed variant calling with samtools. For, some reason > some of the variants have N in ALT column as shown below: > >>>>>> > >>>>>> Chromosome Position SNPid Reference Alternate > QUAL MQ DP > >>>>>> chr21 29989187 . A AN 96.50 60 46 > >>>>>> > >>>>>> This is a homozygous mutation supported by 46 reads with MQ of 60. > >>>>>> > >>>>>> Checked the bam file for this position using mpileup > >>>>>> > >>>>>> samtools mpileup -AB -f ref.fa -r chr21:29989186-29989188 input.bam > >>>>>> > >>>>>> The output is > >>>>>> > >>>>>> chr21 29989187 A 49 > ....,,,,..,,,,+1n,,...,,,,..,,,,,...,,,...,..,,.,.,^]. > 7BF<<FFFB7FF<0FIFIIIFBFIIBFFF<IIIFFB<IIFII7BIBIBB > >>>>>> > >>>>>> Is this a bug in the code that it is called as "AN" insertion. How > should i infer this mutation. > >>>>>> > >>>>>> Any comments from the users of this community are highly valuable. > >>>>>> > >>>>>> > >>>>>> Br > >>>>>> Mehar > >>>>>> > >>>>>> > >>>> > >> > <normal.png><N_ALT.png>------------------------------------------------------------------------------ > >> _______________________________________________ > >> Samtools-help mailing list > >> Samtools-help@lists.sourceforge.net > >> https://lists.sourceforge.net/lists/listinfo/samtools-help > > > > ------------------------------------------------------------------------------ > _______________________________________________ > Samtools-help mailing list > Samtools-help@lists.sourceforge.net > https://lists.sourceforge.net/lists/listinfo/samtools-help >
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