A tiled amplicon protocol for culture-free whole-genome sequencing of M. tuberculosis from clinical specimens.
| Title: | A tiled amplicon protocol for culture-free whole-genome sequencing of M. tuberculosis from clinical specimens. |
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| Authors: | Kalinich CC; Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut, USA.; Gonzalez FL; Department of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut, USA.; Osmaston A; Department of Infection, Immunity, and Inflammation, Institute of Child Health, University College London, London, England.; Universidad Peruana Cayetano Heredia, Lima, Peru.; Breban MI; Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut, USA.; Distefano I; Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut, USA.; Leon C; Universidad Peruana Cayetano Heredia, Lima, Peru.; Coronel J; Universidad Peruana Cayetano Heredia, Lima, Peru.; Tan G; Department of Infection, Immunity, and Inflammation, Institute of Child Health, University College London, London, England.; Crudu V; Institute of Phthisiopneumology, Chisinau, Moldova.; Ciobanu N; Institute of Phthisiopneumology, Chisinau, Moldova.; Codreanu A; Institute of Phthisiopneumology, Chisinau, Moldova.; Solano W; Universidad Peruana Cayetano Heredia, Lima, Peru.; Ráez J; Universidad Peruana Cayetano Heredia, Lima, Peru.; Sheen P; Universidad Peruana Cayetano Heredia, Lima, Peru.; Zimic M; Universidad Peruana Cayetano Heredia, Lima, Peru.; Allicock OM; Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut, USA.; Yale Institute for Global Health, Yale University, New Haven, Connecticut, USA.; Chaguza C; Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut, USA.; Wyllie AL; Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut, USA.; Brandt M; Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut, USA.; Weinberger DM; Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut, USA.; Yale Institute for Global Health, Yale University, New Haven, Connecticut, USA.; Public Health Modeling Unit, Yale School of Public Health, New Haven, Connecticut, USA.; Sobkowiak B; Department of Infection, Immunity, and Inflammation, Institute of Child Health, University College London, London, England.; Public Health Modeling Unit, Yale School of Public Health, New Haven, Connecticut, USA.; Cohen T; Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut, USA.; Public Health Modeling Unit, Yale School of Public Health, New Haven, Connecticut, USA.; Grandjean L; Department of Infection, Immunity, and Inflammation, Institute of Child Health, University College London, London, England.; Universidad Peruana Cayetano Heredia, Lima, Peru.; Grubaugh ND; Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut, USA.; Department of Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut, USA.; Yale Institute for Global Health, Yale University, New Haven, Connecticut, USA.; Public Health Modeling Unit, Yale School of Public Health, New Haven, Connecticut, USA.; Redmond SN; Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, Connecticut, USA.; Yale Institute for Global Health, Yale University, New Haven, Connecticut, USA.; Public Health Modeling Unit, Yale School of Public Health, New Haven, Connecticut, USA. |
| Source: | Journal of clinical microbiology [J Clin Microbiol] 2026 Mar 11; Vol. 64 (3), pp. e0182325. Date of Electronic Publication: 2026 Feb 09. |
| Publication Type: | Journal Article |
| Language: | English |
| Journal Info: | Publisher: American Society for Microbiology Country of Publication: United States NLM ID: 7505564 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1098-660X (Electronic) Linking ISSN: 00951137 NLM ISO Abbreviation: J Clin Microbiol Subsets: MEDLINE |
| Imprint Name(s): | Original Publication: Washington, American Society for Microbiology. |
| MeSH Terms: | Mycobacterium tuberculosis*/genetics ; Mycobacterium tuberculosis*/isolation & purification ; Whole Genome Sequencing*/methods ; Tuberculosis*/microbiology ; Tuberculosis*/diagnosis ; Genome, Bacterial*; Humans |
| Abstract: | Whole-genome sequencing of Mycobacterium tuberculosis can be a valuable tool for TB surveillance and treatment, providing insights into transmission patterns and comprehensive drug susceptibility testing. However, the slow growth of M. tuberculosis means traditional culture-based sequencing methods can take weeks to return results, which has limited the widespread adoption of these techniques and limited their use in clinical decision-making. Tiled amplicon sequencing is a fast, reliable, and cost-effective method of whole-genome sequencing that can be done directly on clinical specimens and has been implemented at scale in academic and public health laboratories across the world; it was the cornerstone of SARS-CoV-2 sequencing and has been adapted for a wide range of viral pathogens. However, similar methods are not yet available for far larger bacterial genomes. Extending this approach to M. tuberculosis would significantly reduce the cost, labor, and turnaround time for whole-genome sequencing. We designed a tiled amplicon panel consisting of 5,128 primers that covers the entire M. tuberculosis genome, the largest tiled amplicon sequencing panel we are aware of to date. Applying our amplicon panels to clinical samples of sputum, we show the ability to recover whole-genome bacterial sequences without the need for culture. The resulting sequence data can be used to determine M. tuberculosis lineage and reliably identify markers of drug resistance. Using this approach in clinical settings could reduce the time needed for comprehensive drug susceptibility testing from weeks to days and enable genomic epidemiology to be performed at scale, even in resource-limited settings.IMPORTANCEWe have developed and tested an amplicon panel, TB-seq, for the priority pathogen Mycobacterium tuberculosis, demonstrating recovery of near-full genomes directly from patient sputum, including mixed and low-concentration samples. This approach significantly reduces the turnaround time for this slow-growing bacterium while maintaining high accuracy in detecting clinically relevant mutations, including those associated with drug resistance. Given the global burden of tuberculosis and the critical need for faster diagnostic solutions, we believe our method has the potential to improve clinical decision-making and public health strategies. |
| Competing Interests: | The authors declare no conflict of interest. |
| Comments: | Update of: bioRxiv. 2024 Dec 20:2024.12.19.629550. doi: 10.1101/2024.12.19.629550.. (PMID: 39763738) |
| Grant Information: | 1S10OD030363-01A1 United States NH NIH HHS; 226007/Z/22/Z United Kingdom WT_ Wellcome Trust; 5R01AI146338-02 United States NH NIH HHS; CK22-2204 United States CC CDC HHS; Medical Student Fellowship United States NH NIH HHS; NU50CK000629 United States CC CDC HHS; PhD Fellowship Great Ormond Street Institute of Child Health; Richard K. Gershon Endowed Medical Student Fellowship Yale University |
| Contributed Indexing: | Keywords: Mycobacterium tuberculosis; amplicon sequencing; genomic epidemiology; pathogen genomics |
| Entry Date(s): | Date Created: 20260209 Date Completed: 20260311 Latest Revision: 20260313 |
| Update Code: | 20260313 |
| PubMed Central ID: | PMC12977623 |
| DOI: | 10.1128/jcm.01823-25 |
| PMID: | 41660836 |
| Database: | MEDLINE |
Journal Article