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

Abstract

The next-generation short-read sequencing technologies that generate comprehensive, whole-genome data with single-nucleotide resolution have already advanced tuberculosis diagnosis, treatment, surveillance and source investigation. Their high costs, tedious and lengthy processes, and large equipment remain major hurdles for research use in high tuberculosis burden countries and implementation into routine care. The portable next-generation sequencing devices developed by Oxford Nanopore Technologies (ONT) are attractive alternatives due to their long-read sequence capability, compact low-cost hardware, and continued improvements in accuracy and throughput. A systematic review of the published literature demonstrated limited uptake of ONT sequencing in tuberculosis research and clinical care. Of the 12 eligible articles presenting ONT sequencing data on at least one Mycobacterium tuberculosis sample, four addressed software development for long read ONT sequencing data with potential applications for M. tuberculosis . Only eight studies presented results of ONT sequencing of M. tuberculosis , of which five performed whole-genome and three did targeted sequencing. Based on these findings, we summarize the standard processes, reflect on the current limitations of ONT sequencing technology, and the research needed to overcome the main hurdles. Summary: The low capital cost, portable nature and continued improvement in the performance of ONT sequencing make it an attractive option for sequencing for research and clinical care, but limited data is available on its application in the tuberculosis field. Important research investment is needed to unleash the full potential of ONT sequencing for tuberculosis research and care.

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

“…Ser315Thr has been widely demonstrated to be a high-confidence and frequent polymorphism that confers an INH-resistance signature of MTB [ 16 ], which was consistently characterized in our study through results of ONT sequencing. It was reported that most INH-resistant isolates harboring the Ser315Thr variant had an MIC of > 5 µg/mL [ 17 ], whereas nucleotide polymorphisms within other INH-resistant isolates with high MIC values have been sparsely investigated.…”

Diagnostic accuracy of nanopore sequencing using respiratory specimens in the diagnosis of pulmonary tuberculosis

“…Owing to its advantages of real-time analysis, updated flow cells, long sequencing reads, reduced error rates, and accessibility of analysis software, nanopore sequencing has revived interest in the clinical application of TB diagnostics ( Dippenaar et al ., 2022 ). A previous study has demonstrated that nanopore sequencing could be a suitable alternative to NGS for the diagnosis of MTB and may be extensively adopted by public health laboratories ( Smith et al ., 2020 ).…”

“…Nanopore sequencing technology, developed by Oxford Nanopore, is a third-generation sequencing technology that is a potential alternative to NGS ( Smith et al ., 2020 ). The nanopore sequencing device is portable, can generate long sequence reads, and has data throughput that is comparable to that of NGS ( Dippenaar et al ., 2022 ). Nanopore sequencing is increasingly being used in TB, with previous studies demonstrating its excellent accuracy in predicting MTB resistance ( Cabibbe et al ., 2020 ;Cervantes et al ., 2020 ); however, its use in early TB screening is limited, particularly using clinical specimens.…”

Global trends of research on tuberculous pleurisy over the past 15 years: A bibliometric analysis

“…Similar studies ( Smith et al., 2020 ) have also shown Nanopore’s competitiveness in the diagnosis of drug-resistant TB. A recent review ( Dippenaar et al., 2022 ) showed that Nanopore’s low cost of sequencing combined with its portability and good performance makes it an attractive option for sequencing in research and clinical care settings. However, data on its application in the field of TB are limited, and there is no research on its application in the diagnosis of TP.…”