The application of Next-Generation Sequencing technology for the detection and diagnosis of non-culturable organisms: viruses and viroids (NGSdetect)

High-throughput sequencing (HTS, formerly also known as next-generation sequencing or pyrosequecing) technologies have seen a tremendous evolution in terms of technical developments. These technologies are more and more used for plant virus discovery, metagenomic and ecological studies but are also increasingly being used in plant virus diagnostic settings, post-entry quarantine investigations or pre-export diagnostics. Conventional virus detection methods based on serology (i.e., enzyme-linked immunosorbent assays) or nucleic acids (polymerase chain reaction (PCR), real-time PCR), requires in depth knowledge for the target pathogen to develop antisera or primers limiting these methods to the detection of mostly known viruses.

The use of HTS for virus detection is not dependent on a priori knowledge about the viruses to be detected which is a major advance in diagnostic testing.

This project aimed to harmonise sample enrichment strategies and HTS workflows for plant virus and viroid detection within a diagnostic framework. Existing bioinformatics approaches were investigated during a proficiency test and a test performance study which demonstrated the power of HTS for diagnostics but also the limitations if diagnosticians are not experienced with the analyses of HTS data. HTS and newly developed sequencing technologies such as Oxford Nanopore Sequencing have a great potential for diagnostics but further work is required to set up guidelines for validation of HTS workflows.