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Targeted Whole Genome Sequencing (TWG-Seq) of Cucumber Green Mottle Mosaic Virus Using Tiled Amplicon Multiplex PCR and Nanopore Sequencing

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Mackie, J., Kinoti, W. M., Chahal, S. I., Lovelock, D. A., Campbell, P. R., Tran-Nguyen, L. T. T., Rodoni, B. C. and Constable, F. E. (2022) Targeted Whole Genome Sequencing (TWG-Seq) of Cucumber Green Mottle Mosaic Virus Using Tiled Amplicon Multiplex PCR and Nanopore Sequencing. Plants, 11 (20). p. 2716. ISSN 2223-7747

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Article Link(s): https://doi.org/10.3390/plants11202716

Publisher URL: https://www.mdpi.com/2223-7747/11/20/2716

Abstract

Rapid and reliable detection tools are essential for disease surveillance and outbreak management, and genomic data is essential to determining pathogen origin and monitoring of transmission pathways. Low virus copy number and poor RNA quality can present challenges for genomic sequencing of plant viruses, but this can be overcome by enrichment of target nucleic acid. A targeted whole genome sequencing (TWG-Seq) approach for the detection of cucumber green mottle mosaic virus (CGMMV) has been developed where overlapping amplicons generated using two multiplex RT-PCR assays are then sequenced using the Oxford Nanopore MinION. Near complete coding region sequences were assembled with ≥100× coverage for infected leaf tissue dilution samples with RT-qPCR cycle quantification (Cq) values from 11.8 to 38 and in seed dilution samples with Cq values 13.8 to 27. Consensus sequences assembled using this approach showed greater than 99% nucleotide similarity when compared to genomes produced using metagenomic sequencing. CGMMV could be confidently detected in historical seed isolates with degraded RNA. Whilst limited access to, and costs associated with second-generation sequencing platforms can influence diagnostic outputs, the portable Nanopore technology offers an affordable high throughput sequencing alternative when combined with TWG-Seq for low copy or degraded samples.

Item Type:Article
Business groups:Horticulture and Forestry Science
Keywords:CGMMV; tiled amplicon sequencing; nanopore
Subjects:Science > Botany > Genetics
Plant culture > Vegetables
Plant pests and diseases
Plant pests and diseases > Plant pathology
Plant pests and diseases > Pest control and treatment of diseases. Plant protection
Deposited On:15 Nov 2022 02:42
Last Modified:15 Nov 2022 02:42

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