BACKGROUND:Phenotypic drug susceptibility testing (pDST) forMycobacterium tuberculosiscan take up to 8 weeks, while conventional molecular tests identify a limited set of resistance mutations. Targeted next-generation sequencing (tNGS) offers rapid results for predicting comprehensive drug resistance, and this study sought to explore its operational feasibility within a public health laboratory in Mumbai, India.METHODS:Pulmonary samples from consenting patients testing Xpert MTB-positive were tested for drug resistance by conventional methods and using tNGS. Laboratory operational and logistical implementation experiences from study team members are shared below.RESULTS:Of the total number of patients tested, 70% (113/161) had no history of previous TB or treatment; however, 88.2% (n= 142) had rifampicin-resistant/multidrug-resistant TB (RR/MDR-TB). There was a high concordance between resistance predictions of tNGS and pDST for most drugs, with tNGS more accurately identifying resistance overall. tNGS was integrated and adapted into the laboratory workflow; however, batching samples caused significantly longer result turnaround time, fastest at 24 days. Manual DNA extraction caused inefficiencies; thus protocol optimisations were performed. Technical expertise was required for analysis of uncharacterised mutations and interpretation of report templates. tNGS cost per sample was US$230, while for pDST this was US$119.CONCLUSIONS:Implementation of tNGS is feasible in reference laboratories. It can rapidly identify drug resistance and should be considered as a potential alternative to pDST.