{
  "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.",
  "abstract_shown": true,
  "affiliations": [
    "The University of Sydney"
  ],
  "article_type": "PUBLISHAHEADOFPRINT",
  "arxiv": null,
  "author_total": 7,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "X. Zhang"
    },
    {
      "name": "E. M. Sim"
    },
    {
      "name": "C. Lam"
    },
    {
      "name": "A. Bustamante"
    },
    {
      "name": "T. Crighton"
    },
    {
      "name": "B. J. Marais"
    },
    {
      "name": "V. Sintchenko"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/b3ge8cxr5e26a.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.09.27.26364088",
  "fulltext_url": "https://www.medrxiv.org/content/10.64898/2026.09.27.26364088v1.full",
  "id": "b3ge8cxr5e26a",
  "issns": [],
  "landing_url": "https://www.medrxiv.org/content/10.64898/2026.09.27.26364088v1",
  "language": null,
  "licence": "CC-BY",
  "licence_label": "CC BY",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "medrxiv",
      "infectious diseases"
    ],
    [
      "openalex",
      "2725|Tuberculosis Research and Epidemiology"
    ]
  ],
  "openalex": "W7218498626",
  "pdf_url": "https://www.medrxiv.org/content/10.64898/2026.09.27.26364088v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-09-27",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/b3ge8cxr5e26a.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:19:37Z",
      "native_id": "10.64898/2026.09.27.26364088",
      "source": "medrxiv",
      "url": "https://www.medrxiv.org/content/10.64898/2026.09.27.26364088v1"
    }
  ],
  "subjects": [
    {
      "field": "medicine",
      "origin": "medrxiv:infectious diseases",
      "subfield": "infectious-disease"
    }
  ],
  "title": "Amplicon-based targeted next generation sequencing (tNGS) for Mycobacterium tuberculosis - unpacking the 'black box'",
  "title_text": "Amplicon-based targeted next generation sequencing (tNGS) for Mycobacterium tuberculosis - unpacking the 'black box'",
  "type": "preprint",
  "updated_at": "2026-10-30T12:19:37Z",
  "url": "https://research.draconitic.com/paper/b3ge8cxr5e26a",
  "venue": "medRxiv",
  "versions": []
}