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Old 03-14-2016, 07:20 AM   #1
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Location: Washington, D.C. metro area

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Default Help with variant calling cell-free DNA data from bam file

My group has been working on a cell free DNA project, library preps done using an assay from Swift BioSci and sequencing on a MiSeq, v2 chemistry 2x151 cycles. We've done sequencing in house, but are still working on getting the recommended pipeline components all up and running (we don't have a bioinformaticist in our group so I'm doing the sequencing and the analysis both).

We have a collaborator that will help us analyze data in the short term and they send us back bam files and variant calls. Variant calls are just in an excel file. When we try to repeat variant calling (because we want the vcf's to do some additional manipulations) from the bam files, it looks like something is breaking and we end up with vcf files that have a lot of entries but the entries all appear to be blank (more or less). For example:

1 134414 . N C,<X> 0 . DP=1;I16=0,0,1,0,0,0,38,1444,0,0,0,0,0,0,16,256;QS=0,1,0;SGB=-0.379885;MQ0F=1 PL 4,3,0,4,3,4

It seems that a part of the problem is that the read depth is only one, so I'm wondering if the reads haven't been grouped yet. The only thing is, the files we're getting back are supposed to have been taken through the entire recommended pipeline so they should be grouped and sorted already, otherwise our collaborator wouldn't have been able to variant call, either. I've tried repeating the bam sort and index and that doesn't appear to help.

So my question is whether the issue I'm having is identifiable to a particular step breaking down (do I need to repeat the read grouping?) or whether I'm doing something wrong? (I believe the latter is more likely, but I'm not sure where to begin troubleshooting my own process.) Any help/info/advice would be greatly appreciated.
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