Development and Comprehensive Evaluation of Culture-Independent, Long Amplicon-Based Targeted Next-Generation Sequencing Methods for Predicting Antimicrobial Resistance in Tuberculosis

species and was highly resistant to interference from other clinical pathogens. To ensure optimal coverage, it is recommended to input at least 500 copies of the genome and sequence 500MB of high-quality FASTQ data. Diagnostic performance evaluations demonstrate that this method achieves 98.35% concordance with phenotypic drug susceptibility testing (pDST) and is consistent with the results obtained from Xpert MTB/RIF assays. The design of long amplicons not only ensures comprehensive coverage of target regions but also simplifies primer design, facilitating compatibility with various sequencing platforms. Compared with previous studies, the optimized ONT tNGS method in this study significantly improves turnaround time, detection accuracy, and the comprehensive coverage of mutations associated with AMR.