Rachelle Bester1, Glynnis Cook2, Johannes H J Breytenbach2, Chanel Steyn2,3, Rochelle De Bruyn1,2, Hans J Maree4,5. 1. Department of Genetics, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa. 2. Citrus Research International, P.O. Box 28, Nelspruit, 1200, South Africa. 3. Department of Plant Pathology, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa. 4. Department of Genetics, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa. hjmaree@sun.ac.za. 5. Citrus Research International, P.O. Box 2201, Matieland, 7602, South Africa. hjmaree@sun.ac.za.
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.
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.
Authors: Guoyan Zhao; Siddharth Krishnamurthy; Zhengqiu Cai; Vsevolod L Popov; Amelia P Travassos da Rosa; Hilda Guzman; Song Cao; Herbert W Virgin; Robert B Tesh; David Wang Journal: PLoS One Date: 2013-10-22 Impact factor: 3.240
Authors: Roberto A Barrero; Kathryn R Napier; James Cunnington; Lia Liefting; Sandi Keenan; Rebekah A Frampton; Tamas Szabo; Simon Bulman; Adam Hunter; Lisa Ward; Mark Whattam; Matthew I Bellgard Journal: BMC Bioinformatics Date: 2017-01-11 Impact factor: 3.169
Authors: Ruvini V Lelwala; Zacharie LeBlanc; Marie-Emilie A Gauthier; Candace E Elliott; Fiona E Constable; Greg Murphy; Callum Tyle; Adrian Dinsdale; Mark Whattam; Julie Pattemore; Roberto A Barrero Journal: Viruses Date: 2022-07-05 Impact factor: 5.818