Targeted Genome Sequencing (TG-Seq) Approaches to Detect Plant Viruses

Abstract

Globally, high-throughput sequencing (HTS) has been used for virus detection in germplasm certification programs. However, sequencing costs have impeded its implementation as a routine diagnostic certification tool. In this study, the targeted genome sequencing (TG-Seq) approach was developed to simultaneously detect multiple (four) viral species of; Pea early browning virus (PEBV), Cucumber mosaic virus (CMV), Bean yellow mosaic virus (BYMV) and Pea seedborne mosaic virus (PSbMV). TG-Seq detected all the expected viral amplicons within multiplex PCR (mPCR) reactions. In contrast, the expected PCR amplicons were not detected by gel electrophoresis (GE). For example, for CMV, GE only detected RNA1 and RNA2 while TG-Seq detected all the three RNA components of CMV. In an mPCR to amplify all four viruses, TG-Seq readily detected each virus with more than 732,277 sequence reads mapping to each amplicon. In addition, TG-Seq also detected all four amplicons within a 10−8 serial dilution that were not detectable by GE. Our current findings reveal that the TG-Seq approach offers significant potential and is a highly sensitive targeted approach for detecting multiple plant viruses within a given biological sample. This is the first study describing direct HTS of plant virus mPCR products. These findings have major implications for grain germplasm healthy certification programs and biosecurity management in relation to pathogen entry into Australia and elsewhere.

The Phylogeography of Potato Virus X Shows the Fingerprints of Its Human Vector

“…To ensure greater reliability of future multiplex RT-PCR detection procedures, we recommend the preparation of new primer sets able to detect the increased PVX, PVS, PVY and PVA sequence diversity revealed by our studies. HTS has proven unsuitable for use in large-scale routine virus detection because of its prohibitive cost and the variable genome structure of RNA viruses, which constitutes a serious barrier to designing diagnostic markers that detect diverse plant virus species [122]. Fortunately, targeted genome sequencing (TC-Seq), an amplicon sequencing strategy involving a multiplex PCR reaction that not only detects diverse virus sequence targets simultaneously, but also greatly reduces cost and workload, holds considerable promise for sensitive, large-scale routine plant virus testing in both biosecurity and healthy stock programs in the future [122].…”

Oxford Nanopore Sequencing Reveals an Exotic Broad bean mottle virus Genome within Australian Grains Post-Entry Quarantine

“…Total RNA was extracted from the positive leaf material using a ZR plant RNA miniprep kit (Zymo Research), and a quality control check was conducted as previously described ( 7 ). The RNA was converted to cDNA using random primers ( 8 ), followed by library preparation using a ligation sequencing kit (SQK-LSK109; ONT); the sequencing was conducted on the MinION platform using a FLO-MIN106D (R9.5) flow cell. Base calling was performed using MinKNOW version 20.10.3 in high accuracy mode.…”

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“…In addition, they are sometimes unable to identify diverse virus variants. Fortunately, the cost of high throughput sequencing has declined rapidly due to advances in sequencing technology, so it is now feasible to use it to overcome such drawbacks and deploy as a routine diagnostic tool for detection of cereal and oilseed viruses in PEQ [338,339].…”