Oxford nanopore sequencing in clinical microbiology and infection diagnostics

Abstract

Extended turnaround times and large economic costs hinder the usage of currently applied screening methods for bacterial pathogen identification (ID) and antimicrobial susceptibility testing. This review provides an overview of current detection methods and their usage in a clinical setting. Issues of timeliness and cost could soon be circumvented, however, with the emergence of detection methods involving single molecule sequencing technology. In the context of bringing diagnostics closer to the point of care, we examine the current state of Oxford Nanopore Technologies (ONT) products and their interaction with third-party software/databases to assess their capabilities for ID and antimicrobial resistance (AMR) prediction. We outline and discuss a potential diagnostic workflow, enumerating (1) rapid sample prep kits, (2) ONT hardware/software and (3) third-party software and databases to improve the cost, accuracy and turnaround times for ID and AMR. Multiple studies across a range of infection types support that the speed and accuracy of ONT sequencing is now such that established ID and AMR prediction tools can be used on its outputs, and so it can be harnessed for near real time, close to the point-of-care diagnostics in common clinical circumstances.

Optimizing Nanopore Sequencing for Rapid Detection of Microbial Species and Antimicrobial Resistance in Patients at Risk of Surgical Site Infections

“…The use of NS in microbial diagnostics is a recent field of enquiry, with the majority of published research proof-of-concept studies that have been published in the last 4 years. There are multiple options for DNA extraction, host DNA depletion, and sequence library preparation, and this has resulted in a lack of standardization with regard to the NS pipeline ( 85 ). As evidenced by this study, protocol design may induce bias with regard to species identification and AMR typing.…”

Rapid and Routine Molecular Typing Using Multiplex Polymerase Chain Reaction and MinION Sequencer

“…In conjunction with consensus sequence generation and homopolymer correction, accurate molecular sequences can be obtained through nanopore sequencing ( Liou et al, 2020 ). MinION sequencer provides the advantages of real-time data analysis, low capital cost, and highly accurate consensus sequence generation, all of which are adequately suited to the constraints of clinical settings ( Sheka et al, 2021 ). Therefore, MinION sequencer has been used in numerous applications of clinical microbiology and infectious diagnostics ( Ma et al, 2013 ; Benitez-Paez et al, 2016 ; Liou et al, 2020 ; Baldan et al, 2021 ; Ben et al, 2021 ; Ferreira et al, 2021 ; Sheka et al, 2021 ; Snell et al, 2021 ; Urban et al, 2021 ).…”

“…MinION sequencer provides the advantages of real-time data analysis, low capital cost, and highly accurate consensus sequence generation, all of which are adequately suited to the constraints of clinical settings ( Sheka et al, 2021 ). Therefore, MinION sequencer has been used in numerous applications of clinical microbiology and infectious diagnostics ( Ma et al, 2013 ; Benitez-Paez et al, 2016 ; Liou et al, 2020 ; Baldan et al, 2021 ; Ben et al, 2021 ; Ferreira et al, 2021 ; Sheka et al, 2021 ; Snell et al, 2021 ; Urban et al, 2021 ). However, the sample size for a MinION flow cell cannot exceed 96 due to the limitations of the barcoding kits.…”

End-User Perspectives on Using Quantitative Real-Time PCR and Genomic Sequencing in the Field

“…Second generation refers to template amplification prior to sequencing and includes Ion Torrent, Pacific Biosciences, and Illumina platforms [9]. Third-generation sequencing such as Oxford Nanopore does not require a template amplification step and offers point-of-care sequencing capability with small portable devices and automated sample preparation capabilities [10,11]. Related to biosurveillance, which would also cover bioforensics and clinical diagnostics, NGS offers an enhanced complement to molecular methods for detection, identification, and characterization of biological threats [12].…”