MutagenesisForge: A Framework for Modeling Codon-Level Mutational Biases and Calculating dN/dS

ABSTRACT The ratio of nonsynonymous (d N ) to synonymous (d S ) substitutions in protein-coding genes is a fundamental metric in molecular evolution to test hypotheses about the relative contributions of genetic drift and natural selection in shaping patterns of protein divergence (Williams et al., 2020). However, interpretation of d N /d S ratios may be confounded by sequence context and specific substitution models (Hughes, 2007; Kryazhimskiy & Plotkin, 2008). We present MutagenesisForge, a modular command-line tool and Python package for simulating codon-level mutagenesis and calculating d N /d S under user-specified conditions. At its core is the MutationModel interface which supports specific substitution matrices and ensures consistency across both Exhaustive and Contextual modes of simulation. These modes allow for users to test evolutionary hypotheses or to generate null distributions of d N /d S across a range of biologically relevant models. As large-scale DNA sequencing data sets continue to be generated both within and between species, MutagenesisForge offers a flexible platform for evolutionary analysis and hypothesis testing of mutational processes in protein-coding genes.