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.

Keywords: MinION; Mycobacterium tuberculosis; Nanopore; amplicon sequencing; drug-resistance; genomics; genotyping.

Fig. 1.

Diagram of the target regions of the gene panel. The lengths of regions are not scaled to genome size.

Fig. 2.

Workflow diagram summarizing the methodology.

Fig. 3.

qPCR of the multiplex PCR product. Each colour represents a different gene. Two replicates per gene and negative controls were included.

Fig. 4.

Depth of coverage by gene in each MinION sequencing run. Genes are ordered by length (from the shortest to the longest). (a) Bar plot representing the median depth by sample and by region of the gene panel. Colours represent the sequencing run and the pattern of the bars represent sample type. (b) Box plot representing the distribution of sequencing depths of each genomic region in the 15 samples. Plots are coloured according to the MinION sequencing run (purple for the first run, blue for the second run).

Fig. 5.

ROC curve used to set the frequency threshold employed to call variants in MinION. Points represent the values for recall and false positive rate obtained when applying different frequency values in MinION variant calling. Both axes are truncated. (a) ROC curve used to set the frequency threshold to call variable SNPs in MinION. Recall and false positive rate value obtained using different variant calling frequency cut-offs for MinION (from 0.1 to 0.9 using increments of 0.1) and comparing with Illumina variant calls at a 0.1 fixed threshold. (b) ROC curve used to determine the frequency threshold to call fixed SNPs in MinION. Both axes are truncated. Recall and false positive rate value obtained using different variant calling frequency cut-offs for MinION (from 0.3 to 0.9 using increments of 0.1) and comparing with Illumina variant calls at a 0.9 fixed threshold. For a detailed result see Fig. S1.

Fig. 6.

Comparison of variants found in Illumina and MinION. (a) Number of variants by sample obtained in MinION and Illumina that passed all quality filters for credible variants defined in Methods. Blue bars represent all variants detected in MinION reads, yellow bars represent variants obtained in Illumina reads, and purple bars represent common variants detected by both sequencing platforms. TP, FP and FN values in the inset box were obtained considering 15 samples. (b) Variant frequency correlation between Illumina and MinION reads. Only SNPs that passed all filters are represented. Points represent variants that appear in both platforms, squares represent discrepant variants only present in MinION reads, and triangles represent variants only present in Illumina. Orange represents phylogenetic variants, purple represents variants associated with antibiotic resistance, and yellow represents other variants. Dashed lines represent the threshold values to define fixed variants for MinION (horizontal) and Illumina (vertical).