Comparison of Sequencing Methods for Obtaining a Whole Genome of Pea Necrotic Yellow Dwarf Virus from U.K. Peas

Abstract

During a project investigating pea viruses in the United Kingdom and developing a workflow for high-throughput sequencing in crop surveillance, pea necrotic yellow dwarf virus (PNYDV) was identified at one site, along with pea enation mosaic virus-1, pea enation mosaic virus-2, pea enation mosaic virus satellite RNA, pea seedborne mosaic virus, and turnip yellows virus. Following an initial finding of small PNYDV contigs by an RNA sequencing protocol on MiSeq (Illumina), confirmation and prevalence testing by PCR and Sanger sequencing determined the average PNYDV prevalence to be 3%. This is the first report of PNYDV, and a nanovirus, in the United Kingdom. Initial characterization of the octopartite genome by PCR revealed that component DNA N had a truncated sequence that differed from those previously reported. This led to confirmation testing using rolling circle amplification (RCA) into MiSeq and then MinION (Oxford Nanopore). Sequences were obtained by both RCA-MiSeq and RCA-MinION, with MinION producing more complete genomes than RCA-MiSeq. Data obtained from sequencing were used to provide an initial estimation of the genomic formula of PNYDV. Previous work has looked into comparing each method; however, this study shows how these methods can be used together. The truncated sequence for DNA N was also found by RCA-MiSeq and RCA-MinION and confirmed by specific primers, but further work is required to understand the impact of this truncation. [Figure: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Keywords

• high-throughput sequencing
• method comparison
• nanovirus
• pea necrotic yellow dwarf virus
• rolling circle amplification