Amplicon-based targeted next generation sequencing (tNGS) for Mycobacterium tuberculosis - unpacking the 'black box'

The WHO recently endorsed two commercial culture-free targeted next-generation sequencing (tNGS) assays for detecting drug resistant Mycobacterium tuberculosis. These proprietary tNGS platforms present a black box. We developed an in-house tNGS assay using Oxford Nanopore Technology sequencing to detect an expanded range of resistance mutations directly from clinical specimens. The assay targeted 44 genomic loci; hsp65 for Mycobacterium tuberculosis complex (MTBC) identification; 12 loci for MTBC lineage classification; and 31 drug resistance loci predicted resistance to 16 anti-TB drugs. In total, 98 amplicons were distributed across three PCR pools. Analytical sensitivity was assessed using M. tuberculosis H37Rv, and proof-of-concept performance was evaluated in 63 archived clinical specimen extracts and one real-life sputum specimen in comparison with Illumina whole genome sequencing (WGS). All amplicons were successfully amplified and sequenced from M. tuberculosis H37Rv genomic DNA. At [≥]100 genome copies/L, 71.4% of amplicons met [≥]20x; mean depth across six replicates, including all loci for rifampicin, ethambutol, pyrazinamide, fluoroquinolone and bedaquiline resistance. In 63 clinical specimens, [≥]90% of amplicons achieved a mean depth of [≥]20x in specimens with IS6110 PCR cycle threshold (Ct) values [≤]24. With Ct value [≤]27 lineage classification was concordant with WGS in 94.7% (18/19) of specimens and drug-susceptibility prediction showed 94.7% (18/19) agreement with both phenotypic and genotypic (WGS) testing. In a single clinical case (Ct 26.5), lineage and resistance-associated mutations were accurately identified directly from sputum. The findings provide new insights into rapid culture-independent tNGS approaches for rapid M. tuberculosis drug resistance detection with lineage assignment.