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Traditional diagnostic methods, such as PCR and mass spectrometry, are limited by species detectability and prolonged culture requirements. To address these limitations, this study introduces a novel approach, named NALC-Seq, which integrates next-generation sequencing (NGS) and target capture sequencing for the direct and comprehensive identification of mycobacteria from sputum samples. A total of 54 patients were prospectively or retrospectively enrolled between April 6, 2021, and November 8, 2022, and sputum samples were subjected to next generation sequencing. The NALC-Seq methodology utilizes custom-designed RNA probes for targeted enrichment of mycobacterial DNA, coupled with species identification via the mlstverse-web system. This system integrates web, upload, head, and cloud nodes to streamline data management and analysis. Our evaluation demonstrated that NALC-Seq achieved high sensitivity (98.1%) and accuracy (83.3%) in detecting diverse mycobacterial species, including rare and drug-resistant subspecies. There were discrepancies with MGIT-Seq in nine samples. These samples exhibited low smear positivity rates, and the detection of environmental mycobacteria suggested potential contamination. Furthermore, real-time identification using the MinION device significantly reduced the turnaround time from 686 to 19 h.These findings highlight the potential of NALC-Seq and mlstverse-web as an integrated diagnostic solution for overcoming the limitations of current methods. The proposed approach offers a rapid, accurate, and comprehensive strategy for mycobacterial identification, with significant implications for clinical diagnostics and public health surveillance.<\/jats:p>","DOI":"10.1007\/s10142-025-01609-x","type":"journal-article","created":{"date-parts":[[2025,5,21]],"date-time":"2025-05-21T05:05:45Z","timestamp":1747803945000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["mlstverse-web: advancing real-time identification of mycobacteria from sputum using targeted sequencing NALC-Seq"],"prefix":"10.1007","volume":"25","author":[{"given":"Yuki","family":"Matsumoto","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kiyoharu","family":"Fukushima","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shunran","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuzy","family":"Fauzyah","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daisuke","family":"Motooka","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haruko","family":"Saito","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"June","family":"Yamauchi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tadayoshi","family":"Nitta","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takuro","family":"Nii","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takanori","family":"Matsuki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazuyuki","family":"Tsujino","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keisuke","family":"Miki","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroshi","family":"Kida","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shota","family":"Nakamura","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,5,21]]},"reference":[{"key":"1609_CR1","doi-asserted-by":"crossref","first-page":"439","DOI":"10.3201\/eid2303.161827","volume":"23","author":"J Adjemian","year":"2017","unstructured":"Adjemian J, Frankland TB, Daida YG, Honda JR, Olivier KN, Zelazny A et al (2017) Epidemiology of nontuberculous mycobacterial lung disease and tuberculosis, Hawaii, USA. 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All the experiments were conducted in accordance with the principles outlined in the Declaration of Helsinki. The ethics boards of Osaka University (31\u20138-3 and 583\u20133) and the National Hospital Organization, Osaka Toneyama Medical Center (TNH-R-2020020) approved this study. Written informed consent was obtained from all the participants.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"The authors declare no competing interests.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"105"}}