Pitfalls of bacterial pan-genome analysis approaches: a case study of <i>Mycobacterium tuberculosis</i> and two less clonal bacterial species
Maximillian G. Marin, Natalia Quinones‐Olvera, Christoph Wippel, Mahboobeh Behruznia, Brendan M. Jeffrey, Michael A. Harris, Brendon Mann, Alex Rosenthal, Karen R. Jacobson, Robin M. Warren, Heng Li, Conor J. Meehan, Maha Farhat
Abstract
SUMMARY: Pan-genome analysis is a fundamental tool for studying bacterial genome evolution; however, the variety in methods used to define and measure the pan-genome poses challenges to the interpretation and reliability of results. Using Mycobacterium tuberculosis, a clonally evolving bacterium with a small accessory genome, as a model system, we systematically evaluated sources of variability in pan-genome estimates. Our analysis revealed that differences in assembly type (short-read versus hybrid), annotation pipeline, and pan-genome software, significantly impact predictions of core and accessory genome size. Extending our analysis to two additional bacterial species, Escherichia coli and Staphylococcus aureus, we observed consistent tool-dependent biases but species-specific patterns in pan-genome variability. Our findings highlight the importance of integrating nucleotide- and protein-level analyses to improve the reliability and reproducibility of pan-genome studies across diverse bacterial populations. AVAILABILITY AND IMPLEMENTATION: Panqc is freely available under an MIT license at https://github.com/maxgmarin/panqc.