OS20 Online 10 December

1. 1EuFMD | Open Session special edition | #OS20se
Andrew E. Shaw, Jemma Wadsworth, Claire Colenutt and Donald P. King
The Pirbright Institute, United Kingdom
Enhanced complete genome sequencing
of foot-and-mouth disease virus using
probe enrichment

2. 2EuFMD | Open Session special edition | #OS20se
Sequencing foot-and-mouth disease using
next generation sequencing (NGS)
Complete genome sequencing enables multiple questions to addressed, for example the
presence of recombination or the fine scale tracing of outbreaks.
‘Deep sequencing’ clinical samples using NGS can obtain complete genomes. However, an
abundance of host nucleic acid and reliance upon good quality RNA limits the sensitivity of
unmodified NGS protocols.
Target enrichment of NGS libraries using probes is a strategy by which to increase the depth of
coverage following sequencing.

3. 3EuFMD | Open Session special edition | #OS20se
Sequencing FMDV using target enrichment
Here, we describe a method employing library of oligonucleotide probes to enrich NGS libraries
prior to sequencing.
In order to mimic clinical samples, we diluted O1 Manisa virus into a negative epithelial
suspension and extracted the RNA. Selected samples were additionally heated for 1h at 60°C.
Based upon real-time RT-PCR, strong, weak and very weak dilutions were selected and
processed for sequencing.
Two libraries were prepared for each sample; one of which was target enriched and the other
remaining as a control.
Libraries were sequenced on the Illumina MiSeq. Sequence data was subjected to reference
alignment against the complete genome of O1 Manisa. Read depth was extracted using
Samtools.

4. 4EuFMD | Open Session special edition | #OS20se
Outcome and summary
Target enrichment dramatically improved the depth of coverage achieved for every sample. For
good quality samples the depth was increased ~100 fold, despite a background of host RNA.
Target enrichment dramatically increased the sensitivity, with a substantial increase in coverage
with weaker samples.
Target enrichment enabled sequence data to be obtained for heat denatured samples, including
a complete capsid sequence.
Target enrichment enabled a complete genome to be obtained using RNA extracted from a swab
taken in an African abattoir.

5. eufmdvirtual.com
8, 10, 15, 17 December 2020
EuFMD / Open Session