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

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.

Figure 1

Read coverage required to obtain an accurate consensus sequence. The consensus sequence resulting from varying levels of coverage was assessed for accuracy. Isolates O/UKG/1450/2001 (blue), O/UKG/1558/2001 (green), O/UKG/1734/2001 (purple), O/UKG/4998/2001 (orange) and O/UKG/14597/2001 (red) alongside the type O exemplar from the serotype panel (black) were analysed. Points on the graph represent a comparison of the identities (scored on the y axis) of a consensus made with total reads and a consensus made with limited read coverage (detailed on the x axis). On average, an identity score of one was maintained up to (and including) a coverage limit of 22 reads. Below this level of coverage, the accuracy of the identities of the compared consensus sequences decreased i.e. consensus sequences made with a depth of 22x reads were identical to the consensus. Sequences created with less than 22x coverage depth were not identical, and therefore considered less accurate.

Figure 2

Application of protocol to field isolates from 2001. Coverage of between 1000–10,000x was achieved for 4/6 UKG 2011 isolates (O/UKG/1450/2001 (blue), O/UKG/1558/2001 (green), O/UKG/1734/2001 (purple) and O/UKG/14597/2001 (red)) with a drop in coverage at the poly(C) tract (~375 bp position). O/UKG/4998/2001 (orange) showed lower coverage of between 10-100x. Primer locations are shown as black arrowheads above the genome illustration.

Figure 3

Genome coverage profiles for FMDV serotype panel. Sequence data coverage at each position along the genome is shown for serotype O (black), A (pink), Asia 1 (orange), C (green), SAT 1 (light blue), SAT 2 (blue), and SAT 3 (yellow). The majority of the coverage is above 1000x. In all viruses tested, a poly(C) tract within the FMDV genome at ~375 bp was associated in a reduction in coverage. The coverage depth observed for SAT 3 was lower than other serotypes. Primer locations are shown as black arrowheads above the genome illustration.

Figure 4

Genome coverage profiles for three non-FMDV panel of viruses. Coverage of 10,000 was achieved for the majority of the EMCV-1 genome (olive). Peaks in coverage can be observed at the location of sequence specific primers used in the RT reaction (~4000 bp and ~8000 bp). A dip in coverage was evident at the poly(C) tract. The ERAV-1 genome showed between 10x and 100x coverage with visible peaks in coverage at the specific primer sites (~4000 bp and ~8000 bp) (black). Approximately 100x coverage of the majority of the VESV-B34 genome was achieved (blue).