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Missing variant using RNA-seq best practices
I am doing variant calling on RNA-seq data processed according to the RNA-seq best practices guide published on this site. In general I am satisfied with the results, but I am missing one variant in a sample that should clearly be there -- not only do I see it on IGV but it's also been detected by targeted DNA sequencing.
When I use either HaplotypeCaller or Mutect2 in a BAM file prior to processing the variant of interest (see picture above, first track on IGV) is found without any issues. However, after STAR 2nd pass, Split'N'Trim and base recalibration (second track), both tools fail to do so; interestingly, other programs like bcftools mpileup + bcftools call do find this variant. I have also tried --disable-tool-default-read-filters and other permissive options in Mutect2, to no avail.
My personal feeling about this is that it has to do something with the clipping almost next to the variant. Indeed, running Mutect2 with various intermediate files reveals the variant stops being detected after the Split'N'Trim step, which introduces the hard clipping.
Is this the expected behaviour? It does not make much sense to me, obviously there will be clipping close to the end of the exon... How to deal with such cases?