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Updated: May 10, 2026
Nanopore DNA Sequencing for Metagenomic Soil Analysis
Published on: December 14, 2017
Rapid Diagnostic Sequencing of Stool DNA Using Targeted Nanopore Sequencing in Patients With a Pulmonary Tuberculosis
Tara Ness1,2, Nontobeko Maphalala3, Wethusonkhe Khumalo3
1Global Tuberculosis Program, Baylor College of Medicine, Houston, Texas, USA.
Background:
Approximately 1.25 million individuals died of tuberculosis in 2023, in part due to ineffective treatment. In patients with paucibacillary tuberculosis, microbiologic confirmation and drug resistance testing via respiratory specimens is challenging; hence, stool samples are increasingly used for microbiologic confirmation. Targeted next-generation sequencing (tNGS) of stool DNA may improve detection of drug-resistant (DR) tuberculosis, helping patients receive appropriate treatments.
Methods:
We assessed the ability of a nanopore tNGS approach using stool to detect drug resistance in a prospective, nested cohort of consecutive participants in Eswatini with pulmonary tuberculosis confirmed via sputum culture or sputum GeneXpert Ultra from 2020 to 2023. We compared stool tNGS with (1) a composite reference standard of diagnostic tools available in the study setting, (2) sputum culture tNGS, and (3) whole-genome sequencing of sputum culture.
Results:
Participants ranged in age from 2 to 80 years (median age, 28 years; interquartile range, 20-40 years), and 45.6% (26 of 57) had human immunodeficiency virus. Based on stool tNGS, 14% of our cohort (8 of 57) had drug resistance, and 8.8% (5 of 57) would have received a different treatment regimen had stool tNGS informed clinical decision making. Stool tNGS with nanopore technology was 94.4% concordant (in 17 of 18) for identifying DR mutations with whole-genome sequencing and identified 90% of resistant mutations (9 of 10) indicated by composite reference standard. Stool tNGS detected resistance not detected by standard methods, including detection of rifampicin resistance associated with rpoB Ile491Phe not detected with culture-based phenotypic drug susceptibility testing (pDST) and GeneXpert Ultra. Stool tNGS also detected bedaquiline, clofazimine, and ethambutol resistance not detected with culture-based pDST in Eswatini. The workflow from stool processing to nanopore tNGS report can be completed in 1 day.
Conclusions:
Stool tNGS of Mycobacterium tuberculosis using nanopore technology provides a rapid and accurate method to inform the design of effective treatment regimens in patients with pulmonary tuberculosis in countries with high DR tuberculosis burdens and limited resources.
