Recommendations from Euphresco projects
The following research project has recently been carried out in the framework of Euphresco (network for phytosanitary research coordination and funding - hosted by EPPO). A report presenting the main objectives and results of this project, as well as recommendations made can be viewed on the Internet.
The application of Next-Generation Sequencing technology for the detection and diagnosis of non-culturable organisms: viruses and viroids (NGSdetect)
High-throughput sequencing (HTS) technologies are increasingly being used in a regulatory context. The project aimed to optimise the application of HTS in diagnostics, and focussed on sample preparation, library preparation, the comparison of different sequencing platforms and bioinformatic analysis. The project brought together many partners with different types of experience, from partners with little or no experience with HTS to experts.
HTS is a powerful technology that enables the simultaneous detection of plant viruses and viroids without a priori knowledge of what may be present. In comparison to other technologies, HTS is still relatively expensive and it should be reserved for critical samples. The outcome of HTS analyses depends on good sample preparation. Various methods exist to enrich viral sequences and, depending on the scope of the analyses, different enrichment methods may be chosen. However, as the sample preparation is crucial for good sequencing results, the best method needs to be empirically validated in each laboratory and adjusted to the particular matrix under analysis. The use of Phaseolus vulgaris endovirus -1 (PvEV-1) is an excellent control for the extraction phase, and can allow users to verify whether a sufficient sequencing depth has been achieved, thus avoiding false negative results.
Another major obstacle in implementing HTS lays in the bioinformatics analyses of HTS data. There is currently no solution that will fit all purposes and therefore, significant bioinformatics expertise is required to correctly interpret HTS data. One potential solution was the use of Virtool that was developed at the Canadian Food Inspection Agency.
Duration of the project: 2016-07-02 to 2019-01-31.
Authors: Ziebell, Heiko; De Jonghe, Kris; Rott, Mike; Nicolaisen, Mogens; Gentit, Pascal; Renvoise, Jean-Philippe; Candresse, Thierry; Fox, Adrian; Varveri, Christina; Melika, George; Krizbai, Laszlo; Angelini, Elisa; Ferretti, Luca; Westenberg, Marcel; Roenhorst, Annelien; Shneyder, Yury; Kornev, Konstantin; Olmos, Antonio; Kreuze, Jan; Ravnikar, Maja; Mehle, Natasa; Maree, Hans J.
Link: https://zenodo.org/record/4467914#.YBK5eOhKiUl
Sources
Euphresco (2021-02). https://www.euphresco.net/projects/ |