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Reference: Application of Targeted Next-Generation Sequencing in the Identification of Drug-Resistant Mycobacterium tuberculosis Clinical cases of MDR and other drug-resistant tuberculosis remains undetected, and current WHO-recommended rapid diagnostics (WRDs) only detect resistance to a limited number of drugs and cover only a single or few resistance-associated gene markers. Targeted next-generation sequencing (tNGS) delivers rapid and simultaneous detection of mutations associated with resistance to tuberculosis across multiple gene targets in a single test. Thus, tNGS offers more comprehensive and precise identification, and drug sensitivity profile including those for new and repurposed drugs which are not covered by current WRDs. Furthermore, tNGS shows high sensitivity and specificity for rifampicin, isoniazid, moxifloxacin, ethambutol, levofloxacin, and pyrazinamide. Eventhough, targeted NGS is costly and technically demanded than WRDs, this is a convenient alternative for prioritized patients requiring comprehensive DST with faster results, or where access to phenotypic DST is limited. In 2024, WHO endorsed tNGS for diagnosing resistance to key first- and second-line drugs.  The targets for tNGS could be amplified using commercial kits, and performed using different platforms like Illumina, Oxford Nanopore Technology and Ion torrent. There are several commercial kits including Deeplex® Myc-TB (GenoScreen, France), AmPORE-TB (Oxford Nanopore Technologies, UK), TBseq® (Hangzhou ShengTing Medical Technology Co., China), DeepChek® 13-Plex KB (ABL Diagnostics, France), and Genes2Me TB NGS Assay (Genes2Me, India). Diverse bio-informatics pipeline options are used for analysis of data generated by the system, and several commercial kits include it’s one analysis pipeline. Literature shows that tNGS has high concordance between phenotypic antimicrobial susceptibility testing (AST) or whole-genome sequencing (WGS), with average agreement exceeding 95%. Furthermore, enhanced detection sensitivity of tNGS than culture or Xpert MTB/RIF assays, especially in culture-negative or low-bacilli samples has been recorded. Reporting rapid turnaround and direct applicability to clinical material without requiring culture are added advantages. In addition to the diagnostics, some commercial tNGS kits offer investigation of moleular epidemiology of MTB strains simultaneously. Thus, tNGS can recreate an essential role in public health efforts, assisting in understanding TB epidemiology, tracking outbreaks, and monitoring drug resistance trends. The World Health Organization recognizes tNGS as a promising complementary tool for genotypic DST and has published conditional implementation guidance, particularly for respiratory samples, reinforcing the article’s relevance in evolving TB diagnostics. However, use of tNGS in the clinical management of patients’ needs further evaluation and efforts. Yet, it is not a near-patient technology, as practical challenges, including access to sequencing infrastructure and standardized interpretation frameworks; especially in low-resource, high-burden TB settings. Since sequencing technologies become more accessible and bioinformatics tools more streamlined, tNGS is poised to play a central role in the global fight against drug-resistant TB in near future.