Amplicon-based targeted next generation sequencing (tNGS) for Mycobacterium tuberculosis - unpacking the 'black box'
- 1The University of Sydney
Abstract
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 [≥]20( 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 (20( 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.
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BibTeX
@misc{zhang2026ampliconbased,
title = {{Amplicon-based targeted next generation sequencing (tNGS) for Mycobacterium tuberculosis - unpacking the 'black box'}},
author = {X. Zhang and E. M. Sim and C. Lam and A. Bustamante and T. Crighton and B. J. Marais and V. Sintchenko},
howpublished = {medRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.27.26364088},
url = {https://www.medrxiv.org/content/10.64898/2026.09.27.26364088v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Amplicon-based targeted next generation sequencing (tNGS) for Mycobacterium tuberculosis - unpacking the 'black box'
AU - X. Zhang
AU - E. M. Sim
AU - C. Lam
AU - A. Bustamante
AU - T. Crighton
AU - B. J. Marais
AU - V. Sintchenko
PY - 2026
DA - 2026/09/27
PB - medRxiv
DO - 10.64898/2026.09.27.26364088
UR - https://www.medrxiv.org/content/10.64898/2026.09.27.26364088v1
AB - 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 [≥]20( 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 (20( 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.
ER -