Tumor heterogeneity (clonal vs subclonal)
A tumor is a mosaic of genetically distinct cell populations; mutations present in every cell (clonal) make far better vaccine targets than those in only a subset (subclonal).
Tumors evolve like a branching tree: early (clonal/truncal) mutations are shared by all tumor cells, while later (subclonal) mutations exist only in particular branches. Sequencing a bulk sample averages over this mixture, and the fraction of cells carrying a mutation can be estimated from variant-allele frequencies.
Clonality is a first-class selection criterion: a vaccine aimed at a clonal neoantigen attacks the whole tumor, whereas a subclonal target leaves most cells untouched and invites escape. Distinguishing clonal from subclonal is itself a noisy inference problem layered on top of variant calling — and a place where better modeling directly improves target choice.