Nanopore Workflow for Grapevine Viroid Surveillance in Kazakhstan: Bypassing rRNA Depletion Through Non-Canonical Priming.

Grapevine ( L.) cultivation is an important agricultural sector worldwide. Its expansion into new areas, like Kazakhstan, brings significant phytosanitary risks. Viroids, such as grapevine yellow speckle viroid 1 (GYSVd-1) and hop stunt viroid (HSVd), are RNA pathogens that threaten vineyard productivity. They can cause a progressive decline through latent infections. Traditional diagnostic methods are usually targeted and therefore not suitable for thorough surveillance. In contrast, modern high-throughput sequencing (HTS) methods often face challenges due to their high costs and complicated sample preparation, such as ribosomal RNA (rRNA) depletion. This study introduces a simplified diagnostic workflow that overcomes these barriers. We utilized the latest Oxford Nanopore V14 cDNA chemistry, which is designed to prevent internal priming, by substituting a targeted oligo(dT)VN priming strategy to facilitate the sequencing of non-polyadenylated viroids from total RNA extracts, completely bypassing the rRNA depletion step and use of random oligonucleotides for c DNA synthesis. This method effectively detects and identifies both GYSVd-1 and HSVd. This workflow significantly reduces the time, cost, and complexity of HTS-based diagnostics. It provides a powerful and scalable tool for establishing strong genomic surveillance and phytosanitary certification programs, which are essential for supporting the growing viticulture industry in Kazakhstan.