Journal Article

High-Throughput Sequencing Methods for the Detection of Two Strawberry Viruses in Post-Entry Quarantine

Khan, Subuhi, Waite, David W., Thompson, Jeremy R., Nunes-Leite, Luciano, Liefting, Lia W.

2024

Overview

High-throughput sequencing (HTS) technologies may be a useful tool for testing imported plant germplasm for multiple pathogens present in a sample, offering strain-generic detection not offered by most PCR-based assays. Metatranscriptomics (RNAseq) and tiled amplicon PCR (TA-PCR) were tested as HTS-based techniques to detect viruses present in low titres. Strawberry mottle virus (SMoV), an RNA virus, and strawberry vein banding virus (SVBV), a DNA virus, were selected for comparison of RNAseq and TA-PCR with quantitative PCR assays. RNAseq of plant ribosomal RNA-depleted samples of low viral titre was used to obtain datasets from 3 M to 120 M paired-end (PE) reads. RNAseq demonstrated PCR-like sensitivity, able to detect as few as 10 viral copies/µL when 60 million (M) PE reads were generated. The custom TA-PCR primer panels designed for each virus were successfully used to recover most of the reference genomes for each virus. Single- and multiple-target TA-PCR allowed the detection of viruses in samples with around 10 viral copies/µL with a minimum continuous sequence length recovery of 500 bp. The limit of detection of the HTS-based protocols described here is comparable to that of quantitative PCR assays. This work lays the groundwork for an increased flexibility in HTS detection of plant viruses.

• Publisher: MDPI AG,MDPI
• Subject: Acids / Control / Design / Diseases and pests / DNA viruses / DNA Viruses - genetics / DNA Viruses - isolation & purification / Fragaria - virology / Genetic aspects / Genome, Viral / Genomes / Germplasm / High-Throughput Nucleotide Sequencing - methods / high-throughput sequencing / Laboratories / Leaves / metatranscriptomics / Methods / Next-generation sequencing / Pathogens / PCR / Plant diseases / Plant Diseases - virology / Plant virus diseases / Plant viruses / Plant Viruses - genetics / Plant Viruses - isolation & purification / Polymerase chain reaction / Polymerase Chain Reaction - methods / Quarantine / RNA sequencing / RNA viruses / RNA Viruses - classification / RNA Viruses - genetics / RNA Viruses - isolation & purification / RNA, Viral - genetics / rRNA / SMoV / Strawberries / SVBV / tiled amplicon sequencing / Viral genetics / Virus diseases of plants / Viruses