EuFMD OS20

1. Introduction
Viral sequence data provides crucial insights into the epidemiology of infectious
diseases such as foot-and-mouth disease (FMD). Oxford Nanopore Technologies’
MinION portable sequencer (Figure 1) is able to generate long reads and produce
real time data which can be used for strain identification, outbreak tracing and
vaccine selection. The cost and size of the sequencer lends itself to use in well-
resourced laboratories and has been used during outbreaks of viral diseases such as
Ebola, Zika and the ongoing COVID-19 pandemic. In this study, we investigated the
suitability of the MinION to characterise FMD virus (FMDV) in clinical samples.
Methods
Laboratory workflow (Figure 2):
A single two-step FMDV universal RT-PCR was used to amplify the capsid-encoding region of three FMDV
serotypes: A, O and Asia 1 from cell culture supernatants (n=3) and clinical samples (tongue epithelium
(n=3) and oral swabs (n=3)). Amplicons were visualised by gel electrophoresis, purified using the QIAquick
PCR Purification Kit (Qiagen) and quantified using a Qubit v3 fluorometer (Invitrogen). Samples were
prepared for sequencing on the MinION and an Illumina MiSeq:
Results Conclusions and future work
The MinION sequencer can characterise FMDV down to strain level in a
variety of different sample matrices and across different serotypes.
Real-time sequencing could be a useful tool for providing information on
circulating strains of FMDV, irrespective of geographical location or virus
pool.
Rapid and accurate information about circulating strains could inform
critical decision making on appropriate use of vaccines to control or prevent
an outbreak of FMD.
Future studies could expand on this work by modifying this lab-based
pipeline for decentralised testing and validating the methods with a larger
number of samples, and for all serotypes of FMDV.
Acknowledgements
This project was funded by Defra (grant code SE2816), BBSRC (grant codes BBS/E/I/00007036 and BBS/E/I/00007037)
and the University of Surrey Vet school.
We also acknowledge the Pirbright High Throughput Sequencing unit (BBS/E/I/00007037), and support through the
Core capability grant (BBS/E/I/00007039).
Thank you to the Transmission Biology, Vesicular Disease Reference Laboratories and the Viral Immunology groups at
The Pirbright Institute for the provision of samples
Data analysis workflow (Figure 3):
Reads basecalled in MinKNOW (v. 4.1.22) and trimmed in EPI2ME based on a qScore of 7 or above.
MinION derived reference genomes for all samples were assembled using Canu (v. 2.0).
Medaka (v. 0.11.5) was used to
generate a polished consensus
sequence
BEDTools (v.2.26.0) was used to
generate coverage profiles.
BioEdit (v.7.2.5) was used to
visually compare consensus
sequences to MiSeq generated
reference sequences. Figure 3: Bioinformatics workflow used in this study (https://github.com/nanoporetech/medaka).
Sample Total reads Mapped
reads (%)
Mean
coverage
depth
Consensus
accuracy
(%)
Sequencing
time to
consensus
Cell culture- Type O 6.23x105 83.62 1.32x105 99.97 10 minutes
Oral swab- Type O 6.37x105 20.24 3.77x104 100.00 2.5 hours
Epithelium- Type O 4.57x105 31.56 2.75x104 100.00 2 minutes
Cell culture- Type A 8.73x105 48.02 8.95x104 100.00 2 minutes
Oral swab- Type A 6.77x105 35.91 4.03x104 99.97 2 minutes
Epithelium- Type A 4.41x105 46.69 3.04x104 100.00 2 minutes
Cell culture- Asia 1 5.16x105 83.30 1.48x105 100.00 2 minutes
Oral swab- Asia 1 7.52x105 81.91 7.47x104 100.00 30 minutes
Epithelium- Asia 1 5.60x105 44.19 3.10x104 100.00 10 minutes
Characterising foot-and-mouth disease virus in
clinical samples using nanopore sequencing
E. Brown1,2 G. Freimanis1 A.Shaw1 D. Horton2 S. Gubbins1 D. King1,3
1) The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey, GU24 0NF. United Kingdom
2) School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Vet School Main Building, Richard Meyjes Road, University of Surrey, Guildford, Surrey, GU2 7AL. United Kingdom.
3) Department of Microbial and Cellular Sciences, School of Biosciences and Medicine, Faculty of Health and Medical Sciences, Stag Hill campus, University of Surrey, Guildford, Surrey, GU2 7XH. United Kingdom.
A range of dsDNA concentrations were tested; cell culture (1204- 1296ng), epithelium (1125- 1281ng) and
oral swabs (400ng-902ng).
Samples were prepared using the ligation (SQK-LSK109) and PCR barcoding kit (EXP-PBC001) from Oxford
Nanopore Technologies.
Reference sequences
were generated on
the MiSeq as per
standard protocols.
Figure 4: The mean coverage per nucleotide for each sample over duration of the
MinION sequencing runs.
All samples achieved a consensus sequence with 100% identity to the MiSeq
generated reference sequences, except for the oral swab infected with type A
and cell culture supernatant infected with type O, where the most accurate
consensus was 99.97% achieved after two and ten minutes of sequencing
respectively (Figure 5). The sequencing time required to reach a consensus
sequence with 100% accuracy varied between samples (Table 1). But, a BLASTn
search of the consensus sequence generated after two minutes revealed the
same phylogenetic placement. The discrepancies between the consensus
sequences and the reference were most commonly at located at
homopolymeric regions consisting of sequential G bases, suggesting the
MinION struggles to sequence GC-rich regions.
Figure 2: Lab-based experimental workflow of the pipeline used in this study
Table 1: The accuracy of the consensus sequences generated the MinION when compared to the MiSeq
reference sequences for each sample and the time taken for the most accurate consensus to be reached
Figure 1: MinION sequencer (image obtained
from Oxford Nanopore Technologies (2019)
https://nanoporetech.com/products/minion)
Amplicon position
Coveragedepthpernucleotide
Figure 5: The mean coverage per nucleotide achieved for all samples when sequenced on the MinION
MinION library
preparation
Sample
MiSeq library preparation
(cell culture)
PCR MinION
sequencing
RNA cDNA
Purified
PCR
product
Raw MinION
data (Fast5)
Inactivation
and RNA
extraction
Reverse
transcription
qRT-PCR
MiSeq sequencing
(reference sequences)
Basecalled reads
(Guppy within
MinKNOW)
Reads aligned to
MinION reference
genomes
(Medaka)
EPI2ME
(QC)
Fast5
files
Reference
genomes
Assembly of
MinION
reference
genomes (Canu)
Polished
consensus
sequences
Comparison with
MiSeq references
Between 70% (cell culture) and 80%
(clinical samples) of reads were under 500
bp in length.
Higher coverage at the first and last 500
bp of the 3’ and 5’ end for all samples
(Figure 4).
Sequencing of all samples recovered
100% of the amplicon length (Figure 4).
The coverage increased exponentially for
all samples and plateaued after 20 hours
of sequencing (Figure 5).
Higher coverage for cell culture derived
samples than clinical material and
generally comparable between oral swabs
and epithelium samples across each time
point, except for Asia 1 (Figure 5)