Plant virome analysis by high-throughput sequencing: concepts and approaches

The metagenomic approach based on high-throughput sequencing is becoming increasingly prevalent for the detection of viral infections in plants. This method allows us to study the species composition of viruses associated with the plant, including novel species, describe their population genetic structure, and develop genetic test systems for routine diagnostics. A metagenomic approach to phytosanitary monitoring can help to determine the cause of unknown plant diseases, which is particularly important for preventing the spread of pathogens, such as viruses. Furthermore, as it is impossible to eliminate plant viruses in field conditions, comprehensive diagnostics using high-throughput sequencing is becoming an effective tool for complying with quarantine regulations on the import of foreign material, as well as for producing high-quality local planting material. High-throughput sequencing is becoming more affordable every year, with both the instrumentation and analytical capacity improving. This review summarizes key approaches to analyzing plant virome using high-throughput sequencing. The analysis process, from sample collection to bioinformatic data processing, validation and interpretation, is described in detail. The features of sequencing platforms and the factors affecting sequencing quality, including contamination, are discussed. Three complementary approaches to processing bioinformatic data are described: mapping reads to reference viral sequences; assembling and annotating contigs; taxonomic classification of reads without assembly. The importance of carefully interpreting the results is emphasized, considering the bioinformatic analysis and the validation by molecular genetic methods. This review will be useful for both researchers and specialists who have no experience with high-throughput sequencing, and those who have used this method for other applications.