Karl J. Romanowicz, Carmen Resnick, Samuel R. Hinton, Calin Plesa
This GitHub repository hosts R Markdown (RMD) files and links to the rendered code for reproducing the data analysis in the following publication:
Romanowicz KJ, Resnick C, Hinton SR, and Plesa C. (2025) Exploring antibiotic resistance in diverse homologs of the dihydrofolate reductase protein family through broad mutational scanning. Science Advances, 11(33), eadw9178. https://www.science.org/doi/10.1126/sciadv.adw9178
Links to Rendered Code:
Broad Mutational Scanning Analysis
Gain-of-Function Mutants Analysis
Resistant Taxa Fitness Analysis
Dialout Variants Fitness Analysis
NCBI BioProject:
- PRJNA1189478 for raw .fastq files used in Makefile
FigShare Repositories:
- DHFR.zip — Contains the input mapping and count files used in the RMarkdown (RMD) analysis pipeline.
- DHFR_Fitness_Data_2025.zip — Provides the processed output files, including fitness data derived from the RMD analysis.
This pipeline processes a library of 1,536 DHFR homologs and their associated mutants, with two-fold redundancy (two codon variants per sequence). Fitness scores are derived from a multiplexed in-vivo assay using a trimethoprim concentration gradient, assessing the ability of these homologs and their mutants to complement functionality in an E. coli knockout strain and their tolerance to trimethoprim treatment. This analysis provides insights into how antibiotic resistance evolves across a range of evolutionary starting points. Sequence data were generated using the Illumina NovaSeq platform with 100 bp paired-end sequencing of amplicons.
Direct any questions regarding this repository to the corresponding author: Calin Plesa.