Characterising Foot-and-Mouth Disease Virus in Clinical Samples Using Nanopore Sequencing

Abstract

The sequencing of viral genomes provides important data for the prevention and control of foot-and-mouth disease (FMD) outbreaks. Sequence data can be used for strain identification, outbreak tracing, and aiding the selection of the most appropriate vaccine for the circulating strains. At present, sequencing of FMD virus (FMDV) relies upon the time-consuming transport of samples to well-resourced laboratories. The Oxford Nanopore Technologies' MinION portable sequencer has the potential to allow sequencing in remote, decentralised laboratories closer to the outbreak location. In this study, we investigated the utility of the MinION to generate sequence data of sufficient quantity and quality for the characterisation of FMDV serotypes O, A, Asia 1. Prior to sequencing, a universal two-step RT-PCR was used to amplify parts of the 5′UTR, as well as the leader, capsid and parts of the 2A encoding regions of FMDV RNA extracted from three sample matrices: cell culture supernatant, tongue epithelial suspension and oral swabs. The resulting consensus sequences were compared with reference sequences generated on the Illumina MiSeq platform. Consensus sequences with an accuracy of 100% were achieved within 10 and 30 min from the start of the sequencing run when using RNA extracted from cell culture supernatants and tongue epithelial suspensions, respectively. In contrast, sequencing from swabs required up to 2.5 h. Together these results demonstrated that the MinION sequencer can be used to accurately and rapidly characterise serotypes A, O, and Asia 1 of FMDV using amplicons amplified from a variety of different sample matrices.

Nanopore Technology and Its Applications in Gene Sequencing

“…Biological nanopores are usually produced by selected bacteria, such as α-hemolysin pore protein [31], MspA from Mycobacterium smegmatis [32], and Phi29 from Bacillus subtilis [33]. These biological nanopores are currently used for disease diagnosis [34], gene sequencing [35], and protein sequencing [36].…”

“…Moreover, it is promising to establish microbe surveillance and response quickly during pandemic, if the nanopore technology can be applied in the area of public health. The representing references are [34,35,67,68] Assemble genomes…”

Rapid metagenomic sequencing for diagnosis and antimicrobial sensitivity prediction of canine bacterial infections

“…The long reads produced by nanopore sequencing also readily facilitate whole genome assembly which potentially allows the linkage of AMR genes to specific bacterial strains [29]. Whilst many previous studies have focussed on human clinical applications, Nanopore sequencing (isolate, metagenomic and 16S) has also already been used in a veterinary context, for example to examine the spread of AMR genes within a veterinary hospital, and to investigate a wide variety of veterinary pathogens [30][31][32][33][34][35][36][37].…”

Comparison of de novo assembly using long-read shotgun metagenomic sequencing of viruses in fecal and serum samples from marine mammals

“…For example, the nanopore-based sequencer MinION can produce read lengths of over 10 kB, which can overcome annotation of genomic repeat regions and structural variations that are difficult to assemble ( Tørresen et al, 2019 ; Hu et al, 2021 ). Nanopore sequencing has been used to detect various viruses from diverse clinical samples ( Quick et al, 2017 ; Hayashida et al, 2019 ; Cohen et al, 2020 ; Brown et al, 2021 ). This technology has been shown to sequence the full genome of four variants of herpes simplex viruses in single read, with read lengths ranging from 100 kb to 2.3 Mb ( Saranathan et al, 2022 ).…”