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Updated: Jan 17, 2026

Amplicon Sequencing using the Long-Read Sequencing Technologies
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Published on: August 29, 2025

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Amplicon Sequencing using the Long-Read Sequencing Technologies.

Morwasehla Modjadji1, Brendon Coenrad Mann1, Johannes Loubser1

  • 1Department of Science and Innovation - National Research Foundation Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University.

Journal of Visualized Experiments : Jove
|September 15, 2025
PubMed
Summary
This summary is machine-generated.

Portable sequencing offers a cost-effective approach for detecting drug-resistant tuberculosis (DR-TB). This method accurately identifies high-frequency resistance mutations but may miss low-frequency variants, impacting tuberculosis diagnostics in resource-limited settings.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Infectious Disease Diagnostics

Background:

  • The World Health Organization (WHO) highlights the need for accessible tuberculosis (TB) diagnostics.
  • Next-generation sequencing (NGS) improves drug-resistant TB (DR-TB) detection, with targeted NGS (tNGS) focusing on resistance mutations.
  • High-cost sequencing platforms limit DR-TB diagnostics in low- and middle-income countries.

Purpose of the Study:

  • To evaluate the utility of portable sequencing for DR-TB detection using a tNGS assay.
  • To compare the performance of portable (long-read) sequencing with high-accuracy (short-read) sequencing for identifying resistance-associated variants in TB.

Main Methods:

  • DNA from rifampicin-resistant TB (RR-TB) isolates was amplified using a tNGS assay.
  • Amplification products were sequenced on both a portable sequencing platform and a high-accuracy short-read platform.
  • Bioinformatics pipelines for long-read and short-read data were utilized for analysis.

Main Results:

  • The portable sequencing approach successfully identified high-frequency resistance-associated variants.
  • Results from portable sequencing correlated well with the reference short-read sequencing platform for common variants.
  • Limitations were observed in detecting low-frequency resistance-associated variants with the portable sequencing method.

Conclusions:

  • Portable sequencing is a viable, cost-effective alternative for DR-TB diagnostics, particularly in resource-limited settings.
  • While effective for high-frequency variants, further optimization is needed to improve sensitivity for low-frequency variants in TB drug resistance detection.