An Illumina-based amplicon sequencing approach designed to determine grapevine fanleaf virus isolates

Traditionally, to comprehensively assess the genetic diversity of a viral isolate, i.e. the population of variants, many molecular steps are necessary, mostly involving PCR amplification, cloning and Sanger sequencing. Lately, with its advent, high throughput sequencing (HTS) has become the method of choice. Both approaches, while extremely informative, may not be economically advantageous to use on a large scale, especially when dealing with highly complex viral populations. Here, we describe an Illumina-based amplicon sequencing (AmpSeq) approach, associated with an analytical workflow for rapid diagnostic, combining HTS quality screening at a more affordable cost. Our multi-primers assay was designed to identify a large diversity of both genomic RNAs (RNA1 and RNA2) of Nepovirus foliumflabelli (also known as Grapevine fanleaf virus, GFLV), compatible with the known genetic diversity of this bipartite virus down to detecting rare variants (i.e. present at less than 2% in an isolate). Beyond diagnostics, our approach can be used and adapted to characterize GFLV populations across vineyards as well as to support the development of GFLV cross-protected vines in France.