I'm trying to establish an analysis pipeline for whole exome seq (WES) of a cancer cell line with acquired resistance. In this experiment we have our cell line grown under normal conditions (WT) and our cell line that has been grown under increasing concentrations of drug and has acquired drug resistance. With three replicates each, we performed WES, and I plan on using Mutect2 through GATK to compare these samples.
In my mind, it makes sense to treat the resistant sample as "Tumor" and the WT sample as "Normal" and call "somatic" mutations using Mutect2. I've read through the Mutect2 tutorial from GATK, and had a few thoughts. Essentially the pipeline is:
1) Create a panel of normals (PON).
2) Run Mutect2 to identify variants.
3) Run GetPileupSummaries and CalculateContamination to account for heterogeneity of tumor samples.
4) Run FilterMutectCalls with the output from (2) and (3).
My biggest question is the necessity of steps 3 and 4. I understand their utility if these were true tumor/normal biopsies, but we don't have any contaminating cell types in these samples, so calculating contamination and filtering based on that doesn't seem necessary. Am I missing something? Does it make sense to take the output of Mutect2 directly and use that as my list of differential mutations?
Thank you for the help!