Rapid and economical drug resistance profiling with Nanopore MinION for clinical specimens with low bacillary burden of Mycobacterium tuberculosis

Abstract

Objective We designed and tested a Nanopore sequencing panel for direct tuberculosis drug resistance profiling. The panel targeted 10 resistance-associated loci. We assessed the feasibility of amplifying and sequencing these loci from 23 clinical specimens with low bacillary burden. Results At least 8 loci were successfully amplified from the majority for predicting first- and second-line drug resistance (14/23, 60.87%), and the 12 specimens yielding all 10 targets were sequenced with Nanopore MinION and Illumina MiSeq. MinION sequencing data was corrected by Nanopolish and recurrent variants were filtered. A total of 67,082 bases across all consensus sequences were analyzed, with 67,019 bases called by both MinION and MiSeq as wildtype. For the 41 single nucleotide variants (SNVs) called by MiSeq with 100% variant allelic frequency (VAF), 39 (95.1%) were called by MinION. For the 22 mixed bases called by MiSeq, a SNV with the highest VAF (70%) was called by MinION. With short assay time, reasonable reagent cost as well as continuously improving sequencing chemistry and signal correction pipelines, this Nanopore method can be a viable option for direct tuberculosis drug resistance profiling in the near future.

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

“…Other studies have also shown 100% of agreement in the prediction of drug-resistance-associated variants between Illumina and MinION Nanopore when variants detected by MinION at an allele frequency below 0.4 are excluded from the analysis [50,51]. Similarly, two recent studies showed complete concordance between both sequencing platforms when applied a threshold 0.8 to call fixed variants [52,53]. Accurate identification of low-frequency variants is relevant for clinical management [11,54] especially for drugs such as fluoroquinolones, for which frequencies ranging from 14-38 % have been reported [55].…”

Nanopore Sequencing for Mycobacterium tuberculosis: a Critical Review of the Literature, New Developments, and Future Opportunities

“…We identified 58 articles of which 12 were eligible. Five articles focused on whole-genome sequencing (WGS) ( 8 , 12 , 21 – 23 ), three on targeted sequencing ( Table 1 ) ( 11 , 24 , 25 ), and four on software development for long-read ONT sequencing data with applications for M. tuberculosis ( Table 2 ) ( 16 , 18 , 19 , 26 ).…”

“…Three studies that assessed ONT sequencing for targeted sequencing of drug resistance loci were all published in 2020 ( 11 , 24 , 25 ). Tafess et al ( 24 ) and Chan et al ( 25 ) used custom-made panels of 19 and 10 drug resistance-associated loci, respectively.…”

“…Three studies that assessed ONT sequencing for targeted sequencing of drug resistance loci were all published in 2020 ( 11 , 24 , 25 ). Tafess et al ( 24 ) and Chan et al ( 25 ) used custom-made panels of 19 and 10 drug resistance-associated loci, respectively. Tafess et al ( 24 ) showed 100% agreement in the detection of drug resistance between ONT and Illumina MiSeq when variants with an allele frequency below 40% reported by ONT sequencing were excluded.…”

Integrative utility of long read sequencing-based whole genome analysis and phenotypic assay on differentiating isoniazid-resistant signature of Mycobacterium tuberculosis

“…Among widely applied platforms, the ONT platform is suitable for sequencing the GC-rich or repetitive proline-glutamate regions within the MTB genome, which might be omitted in results of short-read sequencing [ 14 ]. Moreover, the increased accuracy of the ONT platform of assembling a precise MTB genome is achieved with a higher reading depth compared to that of the Illumina platform [ 7 , 15 ]. The MinION quality score does not equate to the same Phred score, a mean quality score of 20.07 regarding the per base quality score of MinION sequencing were assessed using the CLC genomics workbench in this study.…”