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Updated: Sep 9, 2025

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
MUTAGENESISFORGE: A FRAMEWORK FOR MODELING CODON-LEVEL MUTATIONAL BIASES AND CALCULATING dN/dS
Cooper Koers1, Rob Bierman1, Huixin Xu1
1Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton NJ. 08540, USA.
Abstract:
The ratio of nonsynonymous (dN) to synonymous (dS) 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 dN/dS 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 dN/dS 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 dN/dS 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.
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