Logan, G;
Freimanis, GL;
King, DJ;
Valdazo-González, B;
Bachanek-Bankowska, K;
Sanderson, ND;
Knowles, NJ;
... Cottam, EM; + view all
(2014)
A universal protocol to generate consensus level genome sequences for foot-and-mouth disease virus and other positive-sense polyadenylated RNA viruses using the Illumina MiSeq.
BMC Genomics
, 15
, Article 828. 10.1186/1471-2164-15-828.
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Logan_Universal protocol to generate consensus level genome sequencing for FMDV and other positve sense polyadenylated RNA viruses using the Illumina MiSeq.pdf - Published Version Download (579kB) | Preview |
Abstract
BACKGROUND: Next-Generation Sequencing (NGS) is revolutionizing molecular epidemiology by providing new approaches to undertake whole genome sequencing (WGS) in diagnostic settings for a variety of human and veterinary pathogens. Previous sequencing protocols have been subject to biases such as those encountered during PCR amplification and cell culture, or are restricted by the need for large quantities of starting material. We describe here a simple and robust methodology for the generation of whole genome sequences on the Illumina MiSeq. This protocol is specific for foot-and-mouth disease virus (FMDV) or other polyadenylated RNA viruses and circumvents both the use of PCR and the requirement for large amounts of initial template. RESULTS: The protocol was successfully validated using five FMDV positive clinical samples from the 2001 epidemic in the United Kingdom, as well as a panel of representative viruses from all seven serotypes. In addition, this protocol was successfully used to recover 94% of an FMDV genome that had previously been identified as cell culture negative. Genome sequences from three other non-FMDV polyadenylated RNA viruses (EMCV, ERAV, VESV) were also obtained with minor protocol amendments. We calculated that a minimum coverage depth of 22 reads was required to produce an accurate consensus sequence for FMDV O. This was achieved in 5 FMDV/O/UKG isolates and the type O FMDV from the serotype panel with the exception of the 5′ genomic termini and area immediately flanking the poly(C) region. CONCLUSIONS: We have developed a universal WGS method for FMDV and other polyadenylated RNA viruses. This method works successfully from a limited quantity of starting material and eliminates the requirement for genome-specific PCR amplification. This protocol has the potential to generate consensus-level sequences within a routine high-throughput diagnostic environment.
| Type: | Article |
|---|---|
| Title: | A universal protocol to generate consensus level genome sequences for foot-and-mouth disease virus and other positive-sense polyadenylated RNA viruses using the Illumina MiSeq |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1186/1471-2164-15-828 |
| Publisher version: | http://dx.doi.org/10.1186/1471-2164-15-828 |
| Language: | English |
| Additional information: | Copyright © 2014 Logan et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
| Keywords: | Next generation sequencing, Whole genome sequencing, Foot-and-mouth disease virus, Genome, RNA, virus, FMDV |
| UCL classification: | UCL |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/1521987 |
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