A critical evaluation of Mycobacterium bovis pangenomics, with reference to its utility in outbreak investigation

Abstract

The increased accessibility of next generation sequencing has allowed enough genomes from a given bacterial species to be sequenced to describe the distribution of genes in the pangenome, without limiting analyses to genes present in reference strains. Although some taxa have thousands of whole genome sequences available on public databases, most genomes were sequenced with short read technology, resulting in incomplete assemblies. Studying pangenomes could lead to important insights into adaptation, pathogenicity, or molecular epidemiology, however given the known information loss inherent in analyzing contig-level assemblies, these inferences may be biased or inaccurate. In this study we describe the pangenome of a clonally evolving pathogen, Mycobacterium bovis , and examine the utility of gene content variation in M. bovis outbreak investigation. We constructed the M. bovis pangenome using 1463 de novo assembled genomes. We tested the assumption of strict clonal evolution by studying evidence of recombination in core genes and analyzing the distribution of accessory genes among core monophyletic groups. To determine if gene content variation could be utilized in outbreak investigation, we carefully examined accessory genes detected in a well described M. bovis outbreak in Minnesota. We found significant errors in accessory gene classification. After accounting for these errors, we show that M. bovis has a much smaller accessory genome than previously described and provide evidence supporting ongoing clonal evolution and a closed pangenome, with little gene content variation generated over outbreaks. We also identified frameshift mutations in multiple genes, including a mutation in glpK, which has recently been associated with antibiotic tolerance in Mycobacterium tuberculosis . A pangenomic approach enables a more comprehensive analysis of genome dynamics than is possible with reference-based approaches; however, without critical evaluation of accessory gene content, inferences of transmission patterns employing these loci could be misguided.

Features of Mycobacterium bovis Complete Genomes Belonging to 5 Different Lineages

“…Our analysis showed highly similar genomic features and a conserved synteny within these new 10 complete genomes. Pangenome established with the ten complete genomes and the two other available complete genomes [ 18 , 24 ] confirmed a closed pangenome with a core gene representing 98% of total genes in agreement with previous studies [ 4 , 15 , 57 ]. However, a recent study using the “Get-homologues pipeline” and draft genomes showed an open pangenome and a larger accessory genome in comparison to our study [ 58 , 59 ].…”

“…However, a recent study using the “Get-homologues pipeline” and draft genomes showed an open pangenome and a larger accessory genome in comparison to our study [ 58 , 59 ]. This difference can be explained by the short-read sequences data, which lead to the increase of the accessory genome [ 15 ]. To overcome this problem, Panaroo can be used to clean up annotation errors due to fragmented assemblies or misassembly [ 42 ].…”

“…Indeed, most sequenced genomes are drafts. These genomes are incomplete and contain indels which can bias genetic structure studies or pangenomic studies [ 15 ]. Genome sequencing using long-read technologies now makes it possible to correct these errors and complete the genome at a lower cost [ 16 ].…”

Genetic Features of Mycobacterium avium subsp. paratuberculosis Strains Circulating in the West of France Deciphered by Whole-Genome Sequencing

“…paratuberculosis , Mycobacterium bovis , another major mycobacterial pathogen in cattle but circulating in a multihost system, has genome characteristics of an open pangenome based on the Roary analysis of 70 draft genomes ( 26 ). Recently, a study showed that fragmentation from incomplete genomes severely impacted pangenomic analyses and that Mycobacterium bovis actually possessed a closed pangenome ( 27 ). Our study agrees with the results of a previous report on 316 M. avium subsp.…”

“…It is important to consider that draft genomes introduce a number of problems related to the quality of sequencing data, misassembly, and annotation of fragmentated genomes. All of these problems can influence the size of the pangenome and artificially inflate the size of the accessory genome by the presence of false-positive genes (mainly annotated as “hypothetical protein” genes) and by the absence of certain genes in the draft genomes ( 27 , 29 – 31 ). To avoid as much as possible these biases in our pangenomic analysis, we searched for and excluded redundant gene clusters.…”