A method for in-depth analysis of circular DNA virus populations by unambiguously profiling the low abundant virus variants and partial genomic components.

Severe epidemic outbreaks of diseases associated with newly emerging strains of single-stranded DNA (ssDNA) viruses have led to serious economic losses of numerous important food crops. While the current mitigation strategies are mostly relying on the deployment of genetic resistance in crop varieties, the constantly evolving virus populations have the potential to rapidly break virus resistance. Therefore, the development of diagnostic tools enabling early detection of virus variants associated with hypervirulence and/or expansion to new host species is urgently needed as an effective mitigation solution. Here, we introduce a novel approach by designing a pipeline that allows accurately identifying and characterizing the full-length sequence variants of viral circular DNA genomes utilizing Nanopore sequencing technology and the bioinformatics tool Genome Detective. We demonstrate that the pipeline is suitable to provide an accurate and in-depth analysis of monopartite Tomato yellow leaf curl Sardinia virus (TYLCSV) and multipartite Banana bunchy top virus (BBTV) ssDNA virus populations resulting in the profiling of high- and low-frequency virus variants with ≥1% relative abundance. The approach also enabled the unambiguous detection and characterization of four TYLCSV partial genomic sequences as well as several partial genomic sequences for each BBTV genomic component not previously reported and accumulating during infection.