Analysis of the limited <i>M. tuberculosis</i> accessory genome reveals potential pitfalls of pan-genome analysis approaches

Pan-genome analysis is a fundamental tool for studying bacterial genome evolution; however, the variety of methods used to define and measure the pan-genome poses challenges to the interpretation and reliability of results. To quantify sources of bias and error related to common pan-genome analysis approaches, we evaluated different approaches applied to curated collection of 151 Mycobacterium tuberculosis ( Mtb ) isolates. Mtb is characterized by its clonal evolution, absence of horizontal gene transfer, and limited accessory genome, making it an ideal test case for this study. Using a state-of-the-art graph-genome approach, we found that a majority of the structural variation observed in Mtb originates from rearrangement, deletion, and duplication of redundant nucleotide sequences. In contrast, we found that pan-genome analyses that focus on comparison of coding sequences (at the amino acid level) can yield surprisingly variable results, driven by differences in assembly quality and the softwares used. Upon closer inspection, we found that coding sequence annotation discrepancies were a major contributor to inflated Mtb accessory genome estimates. To address this, we developed panqc, a software that detects annotation discrepancies and collapses nucleotide redundancy in pan-genome estimates. When applied to Mtb and E. coli pan-genomes, panqc exposed distinct biases influenced by the genomic diversity of the population studied. Our findings underscore the need for careful methodological selection and quality control to accurately map the evolutionary dynamics of a bacterial species.

1. 1
   Minimap2: pairwise alignment for nucleotide sequences

   Bioinformatics201816,325 citationsDataRank 1.5
2. 2
   IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era

   Molecular Biology and Evolution202016,005 citationsDataRank 1.5
3. 3
   Twelve years of SAMtools and BCFtools

   GigaScience202115,177 citationsDataRank 1.4
4. 4
   ape 5.0: an environment for modern phylogenetics and evolutionary analyses in R

   Bioinformatics20189,198 citationsDataRank 1.4
5. 5
   NCBI prokaryotic genome annotation pipeline

   Nucleic Acids Research20166,925 citationsDataRank 14.6Top 13%