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

Valencia Region TB Working Group

doi:10.1038/s41467-026-73869-5

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

Valencia Region TB Working Group

Instituto de Biomedicina de Valencia (IBV-CSIC)
University of Valencia
Sequencing and Bioinformatics Genomic Core Unit
European Bioinformatics Institute
University of Oxford
NIHR Oxford Biomedical Research Centre
Hospital Universitari i Politècnic La Fe
Hospital de Denia
Hospital Universitario de La Ribera
Hospital Virgen de los Lírios
Hospital General Universitario de Alicante
Hospital General Universitario de Elche
ISCIII
Hospital Francesc de Borja
Hospital Universitario Doctor Peset
Departamento de Microbiología. Facultad de Medicina. Universidad de Valencia
Consorcio Hospital General Universitario de Valencia
Hospital Clínico Universitario
Hospital General Universitario de Castellón
Consortium for Biomedical Research in Epidemiology and Public Health

Research output: Contribution to journal › Article › peer-review

Abstract

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.

Original language	English
Article number	7242
Number of pages	20
Journal	Nature Communications
Volume	17
Issue number	1
Early online date	6 Jun 2026
DOIs	https://doi.org/10.1038/s41467-026-73869-5
Publication status	E-pub ahead of print - 6 Jun 2026

Data Availability Statement

All data generated or analysed during this study have been deposited in the European Nucleotide Archive under accession codes PRJEB29604, PRJEB38719, PRJEB65844, PRJEB89456, PRJEB89397, PRJEB70424, and PRJEB89421 and are publicly available. The accession numbers for each sample are included in Supplementary Data 1, 2, and 10. Source data are provided with this paper.

Funding

This project received funding from the European Research Council under the European Union’s Horizon 2020 Research and Innovation Programme (Grant 101001038, TB-RECONNECT) awarded to I.C. Additional support was provided by the Spanish Ministry of Science and Innovation (PID2022-137607OB-I00) and the Generalitat Valenciana (Project CIPROM/2023/30), both also awarded to I.C., and PID2021-127919OB-I00 and PID2024-155977OB-I00 to FGC. AGM has been supported by a ‘Formación de Profesorado Universitario’ (FPU) grant (FPU19/04562) from the Spanish Ministry of Universities, and the Health Research 2021 Programme (TB-TARGET_HR21-00415) by LaCaixa Foundation. Work in IC’s laboratory is further supported by the European Union’s Next Generation, through CSIC’s Global Health Platform (PTI Salud Global). JAR is hired under the Generation D initiative, promoted by Red.es, an organisation attached to the Ministry for Digital Transformation and the Civil Service, for the attraction and retention of talent through grants and training contracts (MMT24-PTI-SG-01), financed by the Recovery, Transformation, and Resilience Plan through the European Union’s Next Generation funds.

Funders	Funder number
'la Caixa' Foundation
European Research Council
Ministerio de Ciencia e Innovación	PID2022-137607OB-I00
Horizon 2020 Framework Programme	101001038
Ministerio de Universidades	TB-TARGET_HR21-00415
Civil Service	MMT24-PTI-SG-01
Generalitat Valenciana	PID2024-155977OB-I00, FPU19/04562, PID2021-127919OB-I00, CIPROM/2023/30

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 3 Good Health and Well-being

ASJC Scopus subject areas

General Chemistry
General Biochemistry,Genetics and Molecular Biology
General
General Physics and Astronomy

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10.1038/s41467-026-73869-5Licence: CC BY

https://www.nature.com/articles/s41467-026-73869-5

Cite this

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title = "Complete genomes reveal a refined map of Mycobacterium tuberculosis genetic diversity across evolutionary scales",

abstract = "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. ",

author = "\{Valencia Region TB Working Group\} and Garc{\'i}a-Mar{\'i}n, \{Ana Mar{\'i}a\} and Manuela Torres-Puente and Ll{\'u}cia Martinez-Priego and \{De Marco\}, Griselda and Miguel Moreno-Molina and Martin Hunt and Zamin Iqbal and Ana Gil-Brusola and Oscar Esparcia-Rodr{\'i}guez and Olalla Martinez-Macias and Isabel Escribano and Mar{\'i}a Borr{\'a}s-M{\'a}{\~n}ez and Rodr{\'i}guez, \{Juan Carlos\} and Adelina Gimeno-Gasc{\'o}n and Nieves Gonzalo-Jim{\'e}nez and Ruiz-Garc{\'i}a, \{Mar{\'i}a M.\} and Nieves Orta and Ester Colomer-Roig and Camarena, \{Juan J.\} and Guna-Serrano, \{Mar{\'i}a Remedio\} and Concepci{\'o}n Gimeno and David Navarro and Javier Colomina and Clari, \{Mar{\'i}a Angeles\} and Rafael Borr{\'a}s and L{\'o}pez-Hontangas, \{Jos{\'e} Luis\} and B{\'a}rbara Gomila-Sard and Aurora Blasco and L{\'o}pez, \{Mariana G.\} and Fernando Gonz{\'a}lez-Candelas and Javier Alonso-del-Real and I{\~n}aki Comas",

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doi = "10.1038/s41467-026-73869-5",

language = "English",

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journal = "Nature Communications",

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AU - Valencia Region TB Working Group

AU - García-Marín, Ana María

AU - Torres-Puente, Manuela

AU - Martinez-Priego, Llúcia

AU - De Marco, Griselda

AU - Moreno-Molina, Miguel

AU - Hunt, Martin

AU - Iqbal, Zamin

AU - Gil-Brusola, Ana

AU - Esparcia-Rodríguez, Oscar

AU - Martinez-Macias, Olalla

AU - Escribano, Isabel

AU - Borrás-Máñez, María

AU - Rodríguez, Juan Carlos

AU - Gimeno-Gascón, Adelina

AU - Gonzalo-Jiménez, Nieves

AU - Ruiz-García, María M.

AU - Orta, Nieves

AU - Colomer-Roig, Ester

AU - Camarena, Juan J.

AU - Guna-Serrano, María Remedio

AU - Gimeno, Concepción

AU - Navarro, David

AU - Colomina, Javier

AU - Clari, María Angeles

AU - Borrás, Rafael

AU - López-Hontangas, José Luis

AU - Gomila-Sard, Bárbara

AU - Blasco, Aurora

AU - López, Mariana G.

AU - González-Candelas, Fernando

AU - Alonso-del-Real, Javier

AU - Comas, Iñaki

PY - 2026/6/6

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N2 - 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.

AB - 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.

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DO - 10.1038/s41467-026-73869-5

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SN - 2041-1723

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JO - Nature Communications

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ER -

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

Abstract

Data Availability Statement

Funding

UN SDGs

ASJC Scopus subject areas

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