EvoSubster: a pipeline for evolutionary inference of single- and double-base substitution spectra

Motivation Mutational processes differ widely across the tree of life, yet most existing resources focus on somatic mutations in humans or on a limited set of well-studied species. Results We present EvoSubster, a simple and extensible pipeline for inferring evolutionary single-base and double-base substitution spectra from closely related species using a parsimony-based three-genome comparison. The pipeline automatically downloads NCBI genomes, aligns them, infers substitution direction, quantifies single-base and double-base substitutions, and outputs visualizations. Applying EvoSubster to diverse fungal and cnidarian genomes revealed distinct lineage-specific substitutional signatures, including TTA>TCA and TTA>TGA in mushroom-forming fungi within Agaricomycetes, ACA>AAA and ACG>AAG in cnidarians, CG>TT and GC>AA in Mucoromycota, and frequent A:T-rich adjacent substitutions in Glomeromycetes (arbuscular mycorrhizal fungi). Availability and Implementation EvoSubster is implemented as a set of Python 3, R, and bash scripts and is freely available on GitHub at: https://github.com/marikie/EvoSubster. The pipeline relies on a small number of easy-to-install, publicly available command-line tools. Installation instructions and example workflows are provided in the online documentation.