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

Abstract

A rapid and accurate diagnostic assay represents an important means to detect Mycobacterium tuberculosis , 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 M. tuberculosis 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.

Portable sequencing of Mycobacterium tuberculosis for clinical and epidemiological applications

“…The exclusion of repetitive regions (e.g. pe / ppe genes) led to good agreement between platforms for SNPs and small indels, as previously shown in other work [ 25 ], with discrepancies often being found at an AD between 0.6 and 0.7, suggesting the potential use of the lower cut-off of 0.6 to include more true positive calls. With SNPs covering more than 89% of the genome, an accurate phylogenetic reconstruction was obtained, supporting the utility of ONT for variant identification and lineage profiling.…”

“…This accessibility would be useful for infection control in the high TB transmission setting of the Karonga District, Malawi, the source of our samples. In parallel, the possible direct sequencing from sputum samples has been successfully reported, taking up to 5 days [ 8 , 23 , 25 ], which will shorten the time from specimen collection to a drug-resistance profile, leading to timely and personalized treatment that can be significantly delayed when culture isolation is required (up to 3 weeks).…”

“…Illumina technology with a low sequencing error rate is considered the gold standard and therefore has been applied to inform on drug resistance or transmission, but the performance of ONT, with its known higher error rate, is less clear. Several studies have evaluated the performance of ONT sequencing in target-sequencing approaches for drug-resistance detection [ 25–27 ], finding good concordance between Illumina and ONT, or in WGS analysis [ 28 ]. For ONT sequencing data, an even coverage distribution along the chromosome was observed, with drops coinciding with deleted genes or regions, such as RD152 ( Rv1758c-Rv1765c ) in L2, or insertion sequences, whose presence/absence is variable among different strains.…”

“…It is thus reasonable to expect that the approach will also accurately characterize the sequence of genes that contain variants and, by extension, predict resistance. Previous work has shown accurate drug resistance variant detection through different methods [ 25 , 28 ], with promising results for its use for diagnostics purposes in the clinic. However, for the complete reliance on in silico drug resistance prediction based on genotypes, an improved understanding of catalogue of resistance mutations is essential.…”

MinION Whole-Genome Sequencing in Resource-Limited Settings: Challenges and Opportunities

“…With the continued development of sequencing chemistry and software improvements, the capacity of MinION WGS has improved. MinION WGS has lower base call accuracy during the This article is part of the Topical Collection on Mycology * Kirsten Nielsen knielsen@umn.edu sequencing reaction than first-(Sanger sequencing) and secondgeneration (short-read WGS) sequencing [11,12]. However, various software updates that bioinformatically account for base miscalls have improved the MinION WGS sequencing accuracy to 90% [9].…”