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Pathogens
Volume 14
Issue 8
10.3390/pathogens14080782
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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Article

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

by
Karlygash P. Aubakirova
1,
Zhibek N. Bakytzhanova
1,
Akbota Rakhatkyzy
1,
Laura S. Yerbolova
1,
Natalya P. Malakhova
2 and
Nurbol N. Galiakparov
1,*
1
Laboratory of Biotechnology and Molecular Genetics, M. Aitkhozhin Institute of Molecular Biology and Biochemistry, Almaty 050012, Kazakhstan
2
Laboratory of Plant Bioengineering, M. Aitkhozhin Institute of Molecular Biology and Biochemistry, Almaty 050012, Kazakhstan
*
Author to whom correspondence should be addressed.
Pathogens 2025, 14(8), 782; https://doi.org/10.3390/pathogens14080782
Submission received: 11 July 2025 / Revised: 2 August 2025 / Accepted: 4 August 2025 / Published: 6 August 2025
Versions Notes

Abstract

Grapevine (Vitis vinifera 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.
Keywords: grapevine viroids; hop stunt viroid; grapevine yellow speckle viroid 1; nanopore sequencing; total RNA; viroid detection; Vitis vinifera; Kazakhstan grapevine viroids; hop stunt viroid; grapevine yellow speckle viroid 1; nanopore sequencing; total RNA; viroid detection; Vitis vinifera; Kazakhstan

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MDPI and ACS Style

Aubakirova, K.P.; Bakytzhanova, Z.N.; Rakhatkyzy, A.; Yerbolova, L.S.; Malakhova, N.P.; Galiakparov, N.N. Nanopore Workflow for Grapevine Viroid Surveillance in Kazakhstan: Bypassing rRNA Depletion Through Non-Canonical Priming. Pathogens 2025, 14, 782. https://doi.org/10.3390/pathogens14080782

AMA Style

Aubakirova KP, Bakytzhanova ZN, Rakhatkyzy A, Yerbolova LS, Malakhova NP, Galiakparov NN. Nanopore Workflow for Grapevine Viroid Surveillance in Kazakhstan: Bypassing rRNA Depletion Through Non-Canonical Priming. Pathogens. 2025; 14(8):782. https://doi.org/10.3390/pathogens14080782

Chicago/Turabian Style

Aubakirova, Karlygash P., Zhibek N. Bakytzhanova, Akbota Rakhatkyzy, Laura S. Yerbolova, Natalya P. Malakhova, and Nurbol N. Galiakparov. 2025. "Nanopore Workflow for Grapevine Viroid Surveillance in Kazakhstan: Bypassing rRNA Depletion Through Non-Canonical Priming" Pathogens 14, no. 8: 782. https://doi.org/10.3390/pathogens14080782

APA Style

Aubakirova, K. P., Bakytzhanova, Z. N., Rakhatkyzy, A., Yerbolova, L. S., Malakhova, N. P., & Galiakparov, N. N. (2025). Nanopore Workflow for Grapevine Viroid Surveillance in Kazakhstan: Bypassing rRNA Depletion Through Non-Canonical Priming. Pathogens, 14(8), 782. https://doi.org/10.3390/pathogens14080782

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