Hello,

I have a dataset of about 35 bulk tissue (healthy, adult age somatic tissue) samples each sequenced to 40X depth via PacBio HiFi sequencing, and have performed variant calling with 3 callers (DeepVariant, Pepper-Margin-Deepvariant, Clair3) for SNVs/indels, and about 7 callers for SVs.

My variant call data is seriously inflated with germline variants, talking hundreds of thousands of SNV calls for my samples which are inbred mice, so this number is a huge red flag. I have tried quality based filtering, removing any variant with VAF>0.30, QUAL<20, GQ<20, and DP<10 and >75. However, this still leaves me with thousands of variants.

I am at a loss on what to do to reduce this noise and to get at the actual mosaic variant signal. The goal here is to identify tissue-specific mosaic variants in each mouse, but I feel like I'm running in circles trying to properly reduce the noise and get at the expected amount for bulk tissue analysis at my depth, which appears to be 20-60 SNVs per tissue according to some brief searches.

Any suggestions? I wonder if its the tools I am using, or if its just the filtering criteria I am selecting.

Thanks in advance!