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
| العنوان: | 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 |
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| المؤلفون: | Chanel Steyn, Rachelle Bester, Glynnis Cook, Rochelle de Bruyn, Hans J. Maree, Johannes H. J. Breytenbach |
| المصدر: | Virology Journal, Vol 18, Iss 1, Pp 1-19 (2021) Virology Journal |
| بيانات النشر: | BMC, 2021. |
| سنة النشر: | 2021 |
| مصطلحات موضوعية: | 0106 biological sciences, 0301 basic medicine, Citrus, Viroid, Bioinformatics, Sequence assembly, Computational biology, Biology, 01 natural sciences, Deep sequencing, DNA sequencing, Plant Viruses, lcsh:Infectious and parasitic diseases, 03 medical and health sciences, Citrus tristeza virus, Virology, Reference genes, Human virome, lcsh:RC109-216, Plant Diseases, Research, High-Throughput Nucleotide Sequencing, Ion semiconductor sequencing, Plants, biology.organism_classification, Viroids, 030104 developmental biology, Infectious Diseases, Next-generation sequencing, RNA, CTV, RNA extraction, 010606 plant biology & botany |
| الوصف: | BackgroundHigh-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.MethodsPlant 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.ResultsThe 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.ConclusionsThis 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. |
| اللغة: | English |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::0b11b834751924be09cf85c0d9df50cb https://doaj.org/article/49e28df79d0a4fe9be449fa4c7cb677a |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....0b11b834751924be09cf85c0d9df50cb |
| قاعدة البيانات: | OpenAIRE |
| الوصف غير متاح. |