Enhanced viral detection in grapevine via exome depletion and next‐generation sequencing ( <scp>NGS</scp> )

Abstract Grapevine ( Vitis vinifera L.) is highly prone to viral infections that pose a significant threat to global viticulture sustainability. Traditional detection methods, such as PCR and ELISA, are limited to well‐known pathogens, highlighting the need for more comprehensive and unbiased approaches. Here, we present the development of a cost‐effective viral enrichment system adapted to next‐generation sequencing (NGS) for the detection and characterization of grapevine viruses. Our strategy leverages hybridization‐based capture using biotin‐labelled cDNA probes hereafter named “Chloro‐Zero” designed to selectively deplete highly abundant host transcripts, particularly plastid and ribosomal RNAs while preserving viral RNA. Probe design was informed by transcriptomic analysis of V. vinifera . We evaluated different subtractor‐to‐target RNA ratios, observing a consistent reduction of host RNA and a moderate enrichment of viral sequences. NGS analysis revealed improved recovery of low‐abundance viral transcripts, with coverage levels comparable, to a certain extent, to those obtained using previously available commercial kits, but at a significantly lower cost. Although variability in depletion efficiency was observed, the results demonstrate the potential of this scalable and locally adaptable protocol for virome profiling in grapevines. By addressing key limitations of current depletion methods, our approach facilitates the detection of emerging viral threats and supports the development of more effective certification programs and sustainable management practices. Ongoing improvements in probe design and bioinformatic workflows are expected to enhance performance, providing a robust platform for broader applications in plant virology.