Towards the validation of high-throughput sequencing (HTS) for routine plant virus diagnostics: measurement of variation linked to HTS detection of citrus viruses and viroids
Date
2021-03-22
Journal Title
Journal ISSN
Volume Title
Publisher
BioMed Central
Abstract
Background: High-throughput sequencing (HTS) has been applied successfully for virus and viroid discovery in
many agricultural crops leading to the current drive to apply this technology in routine pathogen detection. The
validation of HTS-based pathogen detection is therefore paramount.
Methods: Plant infections were established by graft inoculating a suite of viruses and viroids from established
sources for further study. Four plants (one healthy plant and three infected) were sampled in triplicate and total RNA
was extracted using two different methods (CTAB extraction protocol and the Zymo Research Quick-RNA Plant Miniprep
Kit) and sent for Illumina HTS. One replicate sample of each plant for each RNA extraction method was also sent
for HTS on an Ion Torrent platform. The data were evaluated for biological and technical variation focussing on RNA
extraction method, platform used and bioinformatic analysis.
Results: The study evaluated the influence of different HTS protocols on the sensitivity, specificity and repeatability
of HTS as a detection tool. Both extraction methods and sequencing platforms resulted in significant differences
between the data sets. Using a de novo assembly approach, complemented with read mapping, the Illumina data
allowed a greater proportion of the expected pathogen scaffolds to be inferred, and an accurate virome profile was
constructed. The complete virome profile was also constructed using the Ion Torrent data but analyses showed that
more sequencing depth is required to be comparative to the Illumina protocol and produce consistent results. The
CTAB extraction protocol lowered the proportion of viroid sequences recovered with HTS, and the Zymo Research kit
resulted in more variation in the read counts obtained per pathogen sequence. The expression profiles of reference
genes were also investigated to assess the suitability of these genes as internal controls to allow for the comparison
between samples across different protocols.
Conclusions: This study highlights the need to measure the level of variation that can arise from the different variables
of an HTS protocol, from sample preparation to data analysis. HTS is more comprehensive than any assay previously
used, but with the necessary validations and standard operating procedures, the implementation of HTS as part
of routine pathogen screening practices is possible.
Description
CITATION: Bester, R., et al. 2021. Towards the validation of high-throughput sequencing (HTS) for routine plant virus diagnostics: measurement of variation linked to HTS detection of citrus viruses and viroids. Virology Journal, 18:61, doi:10.1186/s12985-021-01523-1.
The original publication is available at https://virologyj.biomedcentral.com
Publication of this article was funded by the Stellenbosch University Open Access Fund
The original publication is available at https://virologyj.biomedcentral.com
Publication of this article was funded by the Stellenbosch University Open Access Fund
Keywords
Citrus -- Virus diseases, High-throughput sequencing, Plants -- Diseases and pests -- Biological control, Plants -- Pathogens
Citation
Bester, R., et al. 2021. Towards the validation of high-throughput sequencing (HTS) for routine plant virus diagnostics: measurement of variation linked to HTS detection of citrus viruses and viroids. Virology Journal, 18:61, doi:10.1186/s12985-021-01523-1