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Universal amplification and sequencing of foot-and-mouth disease virus complete genomes using nanopore technology
Andrew E Shaw1, Kebaneilwe Lebani2,3, Lina González Gordon4
1The Pirbright Institute, Ash Road, Pirbright, Surrey, GU24 0NF, UK. andrew.shaw@pirbright.ac.uk.
Background:
Foot-and-mouth disease virus (FMDV) is capable of causing explosive outbreaks among domestic and wild cloven-hoofed animals. Genomic characterisation of FMDV is a crucial component of disease control enabling accurate tracing of disease outbreaks to be undertaken. Nanopore sequencing is an affordable and accessible form of high-throughput sequencing (HTS) technology. However, most published methods for FMDV only sequence genomic fragments or focus upon specific lineages. In this study, a universal FMDV sequencing protocol was developed alongside a bespoke analytical pipeline to sequence any FMDV genome in the absence of prior knowledge regarding the identity of the serotype or lineage.
Methods:
Universal multiplex RT-PCRs were used to amplify overlapping tiles encompassing the entire FMDV genome. The PCR products were pooled and subjected to nanopore sequencing using the portable MinION sequencing device. A bioinformatics pipeline was used to assemble genomes based upon blastn and reference assembly.
Results:
Iterative changes in primer design and pooling resulted in two panels of primers; one set amplifying twenty short fragments (S_scheme), and another set amplifying six longer fragments (L_scheme). Both approaches were shown to be capable of generating FMDV genomes, however the L_scheme was simpler, more reliable and more cost-effective at generating complete genomes. The final L_scheme protocol was assessed using 30 FMDV isolates representing all the currently circulating lineages of FMDV. As part of the development, we successfully trialled the use of this technology in Uganda, a country endemic for FMD.
Conclusions:
The amplification, sequencing and bioinformatics strategy developed here has been assessed using a diverse array of FMDV lineages. Using two multiplex PCR reactions, this approach can successfully generate complete genomes of FMDV in a lineage agnostic fashion. Therefore, the primer sets and approaches described here represent a useful tool for expanding the capacity of laboratories to characterise FMDV at the genomic level.
Insights
A new universal protocol using Nanopore sequencing can now fully characterize Foot-and-mouth disease virus (FMDV) genomes. This method is lineage-agnostic, providing a crucial tool for tracking FMDV outbreaks globally.
Area of Science:
- Virology
- Genomics
- Bioinformatics
Background:
- Foot-and-mouth disease virus (FMDV) causes significant outbreaks in cloven-hoofed animals.
- Genomic characterization is vital for tracing FMDV origins and spread.
- Existing sequencing methods often target specific FMDV lineages or only fragments.
Purpose of the Study:
- To develop a universal Foot-and-mouth disease virus (FMDV) sequencing protocol.
- To create a bioinformatics pipeline for assembling any FMDV genome.
- To enable lineage-agnostic FMDV genome sequencing.
Main Methods:
- Universal multiplex RT-PCRs amplified the entire FMDV genome in overlapping fragments.
- Nanopore sequencing was performed using the MinION device.
- A bioinformatics pipeline utilizing blastn and reference assembly was employed.
Main Results:
- Two primer schemes (S_scheme and L_scheme) were developed, both capable of generating FMDV genomes.
- The L_scheme proved simpler, more reliable, and cost-effective for complete genome generation.
- The protocol was validated on 30 diverse FMDV isolates and trialled in Uganda.
Conclusions:
- The developed strategy successfully generates complete FMDV genomes in a lineage-agnostic manner.
- This approach utilizes two multiplex PCR reactions for broad applicability.
- The primer sets and methods enhance laboratory capacity for genomic characterization of FMDV.
