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Volume 7, Issue 12

Accuracy of an amplicon-sequencing nanopore approach to identify variants in tuberculosis drug-resistance-associated genes Open Access

Abstract

A rapid and accurate diagnostic assay represents an important means to detect , identify drug-resistant strains and ensure treatment success. Currently employed techniques to diagnose drug-resistant tuberculosis include slow phenotypic tests or more rapid molecular assays that evaluate a limited range of drugs. Whole-genome-sequencing-based approaches can detect known drug-resistance-conferring mutations and novel variations; however, the dependence on growing samples in culture, and the associated delays in achieving results, represents a significant limitation. As an alternative, targeted sequencing strategies can be directly performed on clinical samples at high throughput. This study proposes a targeted sequencing assay to rapidly detect drug-resistant strains of using the Nanopore MinION sequencing platform. We designed a single-tube assay that targets nine genes associated with drug resistance to seven drugs and two phylogenetic-determining regions to determine strain lineage and tested it in nine clinical isolates and six sputa. The study’s main aim is to calibrate MinNION variant calling to detect drug-resistance-associated mutations with different frequencies to match the accuracy of Illumina (the current gold-standard sequencing technology) from both culture and sputum samples. After calibrating Nanopore MinION variant calling, we demonstrated 100% agreement between Illumina WGS and our MinION set up to detect known drug resistance and phylogenetic variants in our dataset. Importantly, other variants in the amplicons are also detected, decreasing the recall. We identify minority variants and insertions/deletions as crucial bioinformatics challenges to fully reproduce Illumina WGS results.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): amplicon sequencing , drug-resistance , genomics , genotyping , MinION , Mycobacterium tuberculosis and Nanopore
Funding
This study was supported by the:
  • Conselleria d'Educació, Investigació, Cultura i Esport (Award PROMETEO/2020/012)
    • Principle Award Recipient: ComasIñaki
  • Ministerio de Economía, Indústria y Competividad (Award PID2019-104477RB-I00)
    • Principle Award Recipient: ComasIñaki
  • H2020 European Research Council (Award 101001038)
    • Principle Award Recipient: ComasIñaki
  • H2020 European Research Council (Award 638553)
    • Principle Award Recipient: IñakiComas
© 2021 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution NonCommercial License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2021-12-17
2024-04-20
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Accuracy of an amplicon-sequencing nanopore approach to identify variants in tuberculosis drug-resistance-associated genes
M Gen 7, 000740 (2021); https://doi.org/10.1099/mgen.0.000740
/content/journal/mgen/10.1099/mgen.0.000740
/content/journal/mgen/10.1099/mgen.0.000740
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