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However, existing assays require frequent manual updates to accommodate emerging resistance mutations, limiting scalability and responsiveness.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      We present TOAST (Tuberculosis Optimised Amplicon Sequencing Tool), a novel software tool that automates primer design by integrating mutation frequencies from a curated database of over 68,000 drug-resistant\n                      <jats:italic>M. tuberculosis<\/jats:italic>\n                      genomes. TOAST prioritises regions with the highest clinical relevance, accounting for single-nucleotide polymorphisms, insertions, and deletions. The software supports customisation of design parameters such as amplicon length, melting temperature, and GC content, while screening for undesirable primer properties, including self-dimers and off-target binding. Using TOAST, we designed a multiplex panel of 33 amplicons targeting mutations associated with resistance to 13 anti-TB drugs. These amplicons covered over 97% of resistance mutations in a 68\u00a0K isolate database and were validated using Oxford Nanopore sequencing of two clinical samples, achieving high uniform coverage with a minimum sequencing depth exceeding 50-fold across all targets.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>TOAST represents a major advancement in targeted TB sequencing by integrating large-scale clinical genomic data directly into assay design. This enables rapid, high-coverage, and adaptable amplicon sequencing, enhancing diagnostic precision and surveillance capabilities for drug-resistant TB. TOAST\u2019s framework is also extensible to other pathogens, supporting broader applications in infectious disease genomics.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s12864-025-12247-9","type":"journal-article","created":{"date-parts":[[2025,11,19]],"date-time":"2025-11-19T09:50:04Z","timestamp":1763545804000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["TOAST: a novel tool for designing targeted gene amplicons and an optimised set of primers for high-throughput sequencing in tuberculosis genomic studies"],"prefix":"10.1186","volume":"26","author":[{"given":"Linfeng","family":"Wang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Naphatcha","family":"Thawong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joseph","family":"Thorpe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew","family":"Higgins","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mark Tan Kia","family":"Ik","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Waritta","family":"Sawaengdee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Surakameth","family":"Mahasirimongkol","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jo\u00e3o","family":"Perdig\u00e3o","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Susana","family":"Campino","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taane G.","family":"Clark","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jody E.","family":"Phelan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,11,19]]},"reference":[{"key":"12247_CR1","unstructured":"Global Tuberculosis Report. 2023 [Internet]. 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Available from:\u00a0https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7512154\/.","DOI":"10.1128\/JCM.01582-19"}],"container-title":["BMC Genomics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s12864-025-12247-9.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1186\/s12864-025-12247-9","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s12864-025-12247-9.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,16]],"date-time":"2026-06-16T13:12:41Z","timestamp":1781615561000},"score":1,"resource":{"primary":{"URL":"https:\/\/link.springer.com\/10.1186\/s12864-025-12247-9"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,19]]},"references-count":21,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["12247"],"URL":"https:\/\/doi.org\/10.1186\/s12864-025-12247-9","relation":{},"ISSN":["1471-2164"],"issn-type":[{"value":"1471-2164","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11,19]]},"assertion":[{"value":"5 June 2025","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"17 October 2025","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"19 November 2025","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"This study did not involve the collection of new human or animal data. Two clinical DNA samples were obtained from TB patients who provided informed consent, as part of studies approved by the Ethics Committee for Human Research at the Faculdade de Farm\u00e1cia, Universidade de Lisboa. The 68\u00a0K isolate database was compiled from publicly available sequence data sourced from the ENA. All analyses were conducted on de-identified and publicly available datasets, with all personal identifiers removed in compliance with relevant ethical standards. This work was performed in accordance with the principles of the Declaration of Helsinki.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare no competing interests.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"1058"}}