Complete genomes reveal a refined map of Mycobacterium tuberculosis genetic diversity across evolutionary scales

Elucidating the evolution and epidemiology of Mycobacterium tuberculosis requires comprehensive characterization of its genomic diversity; however, short-read sequencing fails to resolve part of this variation. Here, we assembled 216 complete genomes from clinical isolates in the Valencia Region, Spain, using long-read sequencing. This dataset, mostly encompassing Lineage 4, provides a refined map of M. tuberculosis genetic diversity across evolutionary scales. Complete genomes uncover a median of 312 (-1 to 792) additional SNPs per pairwise comparison, revealing an estimated evolutionary rate 1.44-fold higher than that inferred from short-read mapping. This diversity is concentrated in discrete hotspots, particularly within the pe/ppe gene family, where gene conversion is a major driver of nucleotide diversity. While most PE/PPE epitopes remain highly conserved, suggesting strong purifying selection, some involved in vaccine candidates are affected by gene conversion, with unknown consequences. At the epidemiological scale, additional resolution is gained from SNPs previously masked and newly resolved indels and structural variation, refining genetic transmission networks. Finally, at the within-host level, the use of patient-specific reference genomes allows us to capture genuine diversity during infection, showing that previous approaches led to false positive calls. Together, these findings delineate the landscape of M. tuberculosis genomic diversity and provide a framework for more accurate inference of pathogen evolution, host-pathogen interactions, and transmission dynamics.