vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/vjgb-26-35vavilov-5048Research ArticleВЫСОКОПРОИЗВОДИТЕЛЬНОЕ СЕКВЕНИРОВАНИЕHIGH-THROUGHPUT SEQUENCINGАнализ вирома растений с помощью высокопроизводительного секвенирования: принципы и подходыPlant virome analysis by high-throughput sequencing: concepts and approachesБелкинаД. Д.BelkinaD. D.

Москва

Moscow

coatprotein@bk.ruВиноградоваС. В.VinogradovaS. V.

Москва

Moscow

Федеральный исследовательский центр «Фундаментальные основы биотехнологии» Российской академии наукРоссияFederal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of SciencesRussian Federation202606042026302311320Copyright © Белкина Д.Д., Виноградова С.В., 20262026Белкина Д.Д., Виноградова С.В.Belkina D.D., Vinogradova S.V.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/5048

В последние годы метагеномный подход, основанный на высокопроизводительном секвенировании, находит все большее применение в диагностике вирусных инфекций растений. Этот метод позволяет изучить видовой состав вирусов, ассоциированных с исследуемым растением, и в том числе обнаружить ранее не описанные виды, охарактеризовать их популяционно-генетическую структуру и разработать генетические тестсистемы для рутинной диагностики. Проведение фитосанитарного мониторинга с использованием метагеномного подхода может способствовать определению этиологии неизвестных заболеваний растений, что особенно важно для предотвращения распространения таких патогенов, как вирусы. Кроме того, с учетом невозможности элиминации вирусов растений в полевых условиях комплексная диагностика с помощью высокопроизводительного секвенирования становится эффективным инструментом как в целях соблюдения карантинного законодательства при ввозе импортного материала, так и для получения отечественного посадочного материала высоких категорий. При этом с каждым годом высокопроизводительное секвенирование становится более доступным: расширяется как приборно-техническая, так и аналитическая база. В настоящем обзоре систематизированы ключевые подходы к анализу вирома растений с помощью высокопроизводительного секвенирования. В основной части статьи описаны этапы проведения анализа: от сбора образцов до биоинформатической обработки данных, ее валидации и интерпретации. Подробно рассмотрены особенности современных платформ секвенирования и факторы, влияющие на качество чтения, в том числе контаминация. Охарактеризованы три взаимодополняющих подхода для обработки биоинформатических данных: картирование чтений на референсные последовательности вирусов; сборка и аннотация контигов; таксономическая классификация чтений без дополнительной сборки. Особое внимание уделено необходимости тщательной интерпретации результатов с учетом как биоинформатического анализа, так и валидации идентификации молекулярно-генетическими методами. Обзор будет полезен как для исследователей и специалистов, не имеющих опыта работы с высокопроизводительным секвенированием, так и для тех, кто использовал этот инструмент для выполнения других задач.

The metagenomic approach based on high-throughput sequencing is becoming increasingly prevalent for the detection of viral infections in plants. This method allows us to study the species composition of viruses associated with the plant, including novel species, describe their population genetic structure, and develop genetic test systems for routine diagnostics. A metagenomic approach to phytosanitary monitoring can help to determine the cause of unknown plant diseases, which is particularly important for preventing the spread of pathogens, such as viruses. Furthermore, as it is impossible to eliminate plant viruses in field conditions, comprehensive diagnostics using high-throughput sequencing is becoming an effective tool for complying with quarantine regulations on the import of foreign material, as well as for producing high-quality local planting material. High-throughput sequencing is becoming more affordable every year, with both the instrumentation and analytical capacity improving. This review summarizes key approaches to analyzing plant virome using high-throughput sequencing. The analysis process, from sample collection to bioinformatic data processing, validation and interpretation, is described in detail. The features of sequencing platforms and the factors affecting sequencing quality, including contamination, are discussed. Three complementary approaches to processing bioinformatic data are described: mapping reads to reference viral sequences; assembling and annotating contigs; taxonomic classification of reads without assembly. The importance of carefully interpreting the results is emphasized, considering the bioinformatic analysis and the validation by molecular genetic methods. This review will be useful for both researchers and specialists who have no experience with high-throughput sequencing, and those who have used this method for other applications.

метагеномикавысокопроизводительное секвенированиевирусывиром растенийбиоинформатикаmetagenomicshigh-throughput sequencingvirusesplant viromebioinformaticsThis work was supported by Russian Science Foundation grant No. 23-16-00232.ReferencesAtallah S.S., Gómez M.I., Fuchs M.F., Martinson T.E. Economic impact of grapevine leafroll disease on Vitis vinifera cv. Cabernet Franc in Finger Lakes vineyards of New York. Am J Enol Vitic. 2012;63(1): 73-79. doi 10.5344/ajev.2011.11055Atallah S.S., Gómez M.I., Fuchs M.F., Martinson T.E. Economic impact of grapevine leafroll disease on Vitis vinifera cv. Cabernet Franc in Finger Lakes vineyards of New York. Am J Enol Vitic. 2012;63(1): 73-79. doi 10.5344/ajev.2011.11055Baizan-Edge A., Cock P., MacFarlane S., McGavin W., Torrance L., Jones S. Kodoja: a workflow for virus detection in plants using k-mer analysis of RNA-sequencing data. J Gen Virol. 2019;100(3):533-542. doi 10.1099/jgv.0.001210Baizan-Edge A., Cock P., MacFarlane S., McGavin W., Torrance L., Jones S. Kodoja: a workflow for virus detection in plants using k-mer analysis of RNA-sequencing data. J Gen Virol. 2019;100(3):533-542. doi 10.1099/jgv.0.001210Bankevich A., Nurk S., Antipov D., Gurevich A.A., Dvorkin M., Kulikov A.S., Lesin V.M., … Sirotkin A.V., Vyahhi N., Tesler G., Alekseyev M.A., Pevzner P.A. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19(5):455-477. doi 10.1089/cmb.2012.0021Bankevich A., Nurk S., Antipov D., Gurevich A.A., Dvorkin M., Kulikov A.S., Lesin V.M., … Sirotkin A.V., Vyahhi N., Tesler G., Alekseyev M.A., Pevzner P.A. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19(5):455-477. doi 10.1089/cmb.2012.0021Belkina D., Karpova D., Porotikova E., Lifanov I., Vinogradova S. Grapevine virome of the Don ampelographic collection in Russia has concealed five novel viruses. Viruses. 2023;15(12):2429. doi 10.3390/v15122429Belkina D., Karpova D., Porotikova E., Lifanov I., Vinogradova S. Grapevine virome of the Don ampelographic collection in Russia has concealed five novel viruses. Viruses. 2023;15(12):2429. doi 10.3390/v15122429Bolger A.M., Lohse M., Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114-2120. doi 10.1093/bioinformatics/btu170Bolger A.M., Lohse M., Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114-2120. doi 10.1093/bioinformatics/btu170Buchfink B., Xie C., Huson D.H. Fast and sensitive protein alignment using DIAMOND. Nat Methods. 2015;12(1):59-60. doi 10.1038/nmeth.3176Buchfink B., Xie C., Huson D.H. Fast and sensitive protein alignment using DIAMOND. Nat Methods. 2015;12(1):59-60. doi 10.1038/nmeth.3176Camacho C., Coulouris G., Avagyan V., Ma N., Papadopoulos J., Bealer K., Madden T.L. BLAST+: architecture and applications. BMC Bioinformatics. 2009;10(1):421. doi 10.1186/1471-2105-10-421Camacho C., Coulouris G., Avagyan V., Ma N., Papadopoulos J., Bealer K., Madden T.L. BLAST+: architecture and applications. BMC Bioinformatics. 2009;10(1):421. doi 10.1186/1471-2105-10-421Chevreux B., Wetter T., Suhai S. Genome sequence assembly using trace signals and additional sequence information. In: Proceedings of the German Conference on Bioinformatics (GCB) 99. Hannover, 1999;45-56Chevreux B., Wetter T., Suhai S. Genome sequence assembly using trace signals and additional sequence information. In: Proceedings of the German Conference on Bioinformatics (GCB) 99. Hannover, 1999;45-56Chin C.-S., Peluso P., Sedlazeck F.J., Nattestad M., Concepcion G.T., Clum A., Dunn C., … Luo C., Ecker J.R., Cantu D., Rank D.R., Schatz M.C. Phased diploid genome assembly with single-molecule real-time sequencing. Nat Methods. 2016;13(12):1050-1054. doi 10.1038/nmeth.4035Chin C.-S., Peluso P., Sedlazeck F.J., Nattestad M., Concepcion G.T., Clum A., Dunn C., … Luo C., Ecker J.R., Cantu D., Rank D.R., Schatz M.C. Phased diploid genome assembly with single-molecule real-time sequencing. Nat Methods. 2016;13(12):1050-1054. doi 10.1038/nmeth.4035Cholet F., Ijaz U.Z., Smith C.J. Reverse transcriptase enzyme and priming strategy affect quantification and diversity of environmental transcripts. Environ Microbiol. 2020;22(6):2383-2402. doi 10.1111/1462-2920.15017Cholet F., Ijaz U.Z., Smith C.J. Reverse transcriptase enzyme and priming strategy affect quantification and diversity of environmental transcripts. Environ Microbiol. 2020;22(6):2383-2402. doi 10.1111/1462-2920.15017Cobbin J.C., Charon J., Harvey E., Holmes E.C., Mahar J.E. Current challenges to virus discovery by meta-transcriptomics. Curr Opin Virol. 2021;51:48-55. doi 10.1016/j.coviro.2021.09.007Cobbin J.C., Charon J., Harvey E., Holmes E.C., Mahar J.E. Current challenges to virus discovery by meta-transcriptomics. Curr Opin Virol. 2021;51:48-55. doi 10.1016/j.coviro.2021.09.007Crossley B.M., Bai J., Glaser A., Maes R., Porter E., Killian M.L., Clement T., Toohey-Kurth K. Guidelines for Sanger sequencing and molecular assay monitoring. J Vet Diagn Invest. 2020;32(6):767-775. doi 10.1177/1040638720905833Crossley B.M., Bai J., Glaser A., Maes R., Porter E., Killian M.L., Clement T., Toohey-Kurth K. Guidelines for Sanger sequencing and molecular assay monitoring. J Vet Diagn Invest. 2020;32(6):767-775. doi 10.1177/1040638720905833Dobin A., Gingeras T.R. Mapping RNA-seq reads with STAR. Curr Protoc Bioinformatics. 2015;51(1):11.14.1-11.14.19. doi 10.1002/0471250953.bi1114s51Dobin A., Gingeras T.R. Mapping RNA-seq reads with STAR. Curr Protoc Bioinformatics. 2015;51(1):11.14.1-11.14.19. doi 10.1002/0471250953.bi1114s51Fall M.L., Xu D., Lemoyne P., Clément G., Moffett P., Ritzenthaler C. An innovative binding-protein-based dsRNA extraction method: comparison of cost-effectiveness of virus detection methods using high-throughput sequencing. Mol Ecol Resour. 2025;25(7):e14111. doi 10.1111/1755-0998.14111Fall M.L., Xu D., Lemoyne P., Clément G., Moffett P., Ritzenthaler C. An innovative binding-protein-based dsRNA extraction method: comparison of cost-effectiveness of virus detection methods using high-throughput sequencing. Mol Ecol Resour. 2025;25(7):e14111. doi 10.1111/1755-0998.14111Filloux D., Dallot S., Delaunay A., Galzi S., Jacquot E., Roumagnac P. Metagenomics approaches based on virion-associated nucleic acids (VANA): an innovative tool for assessing without a priori viral diversity of plants. In: Lacomme C. (Ed.) Plant Pathology. Methods in Molecular Biology. Vol. 1302. New York: Humana Press, 2015; 249-257. doi 10.1007/978-1-4939-2620-6_18Filloux D., Dallot S., Delaunay A., Galzi S., Jacquot E., Roumagnac P. Metagenomics approaches based on virion-associated nucleic acids (VANA): an innovative tool for assessing without a priori viral diversity of plants. In: Lacomme C. (Ed.) Plant Pathology. Methods in Molecular Biology. Vol. 1302. New York: Humana Press, 2015; 249-257. doi 10.1007/978-1-4939-2620-6_18Fitzpatrick A.H., Rupnik A., O’Shea H., Crispie F., Keaveney S., Cotter P. High throughput sequencing for the detection and characterization of RNA viruses. Front Microbiol. 2021;12:621719. doi 10.3389/fmicb.2021.621719Fitzpatrick A.H., Rupnik A., O’Shea H., Crispie F., Keaveney S., Cotter P. High throughput sequencing for the detection and characterization of RNA viruses. Front Microbiol. 2021;12:621719. doi 10.3389/fmicb.2021.621719Gaafar Y.Z.A., Ziebell H. Comparative study on three viral enrichment approaches based on RNA extraction for plant virus/viroid detection using high-throughput sequencing. PLoS One. 2020;15(8): e0237951. doi 10.1371/journal.pone.0237951Gaafar Y.Z.A., Ziebell H. Comparative study on three viral enrichment approaches based on RNA extraction for plant virus/viroid detection using high-throughput sequencing. PLoS One. 2020;15(8): e0237951. doi 10.1371/journal.pone.0237951Gallo Y., Marín M., Gutiérrez P. Detection of RNA viruses in Solanum quitoense by high-throughput sequencing (HTS) using total and double stranded RNA inputs. Physiol Mol Plant Pathol. 2021;113: 101570. doi 10.1016/j.pmpp.2020.101570Gallo Y., Marín M., Gutiérrez P. Detection of RNA viruses in Solanum quitoense by high-throughput sequencing (HTS) using total and double stranded RNA inputs. Physiol Mol Plant Pathol. 2021;113: 101570. doi 10.1016/j.pmpp.2020.101570Han Y., He J., Li M., Peng Y., Jiang H., Zhao J., Li Y., Deng F. Unlocking the potential of metagenomics with the PacBio high-fidelity sequencing technology. Microorganisms. 2024;12(12):2482. doi 10.3390/microorganisms12122482Han Y., He J., Li M., Peng Y., Jiang H., Zhao J., Li Y., Deng F. Unlocking the potential of metagenomics with the PacBio high-fidelity sequencing technology. Microorganisms. 2024;12(12):2482. doi 10.3390/microorganisms12122482Hu X., Hurtado-Gonzales O.P., Adhikari B.N., French-Monar R.D., Malapi M., Foster J.A., McFarland C.D. PhytoPipe: a phytosanitary pipeline for plant pathogen detection and diagnosis using RNA-seq data. BMC Bioinformatics. 2023;24(1):470. doi 10.1186/s12859-023-05589-2Hu X., Hurtado-Gonzales O.P., Adhikari B.N., French-Monar R.D., Malapi M., Foster J.A., McFarland C.D. PhytoPipe: a phytosanitary pipeline for plant pathogen detection and diagnosis using RNA-seq data. BMC Bioinformatics. 2023;24(1):470. doi 10.1186/s12859-023-05589-2Javaran V.J., Moffett P., Lemoyne P., Xu D., Adkar-Purushothama C.R., Fall M.L. Grapevine virology in the third-generation sequencing era: from virus detection to viral epitranscriptomics. Plants. 2021; 10(11):2355. doi 10.3390/plants10112355Javaran V.J., Moffett P., Lemoyne P., Xu D., Adkar-Purushothama C.R., Fall M.L. Grapevine virology in the third-generation sequencing era: from virus detection to viral epitranscriptomics. Plants. 2021; 10(11):2355. doi 10.3390/plants10112355Jones R.A.C. Global plant virus disease pandemics and epidemics. Plants. 2021;10(2):233. doi 10.3390/plants10020233Jones R.A.C. Global plant virus disease pandemics and epidemics. Plants. 2021;10(2):233. doi 10.3390/plants10020233Kim D., Song L., Breitwieser F.P., Salzberg S.L. Centrifuge: rapid and sensitive classification of metagenomic sequences. Genome Res. 2016;26(12):1721-1729. doi 10.1101/gr.210641.116Kim D., Song L., Breitwieser F.P., Salzberg S.L. Centrifuge: rapid and sensitive classification of metagenomic sequences. Genome Res. 2016;26(12):1721-1729. doi 10.1101/gr.210641.116Kim D., Paggi J.M., Park C., Bennett C., Salzberg S.L. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019;37(8):907-915. doi 10.1038/s41587-019-0201-4Kim D., Paggi J.M., Park C., Bennett C., Salzberg S.L. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019;37(8):907-915. doi 10.1038/s41587-019-0201-4Koren S., Walenz B.P., Berlin K., Miller J.R., Bergman N.H., Phillippy A.M. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 2017; 27(5):722-736. doi 10.1101/gr.215087.116Koren S., Walenz B.P., Berlin K., Miller J.R., Bergman N.H., Phillippy A.M. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 2017; 27(5):722-736. doi 10.1101/gr.215087.116Kurochkin V.E., Alekseev Y.I., Petrov D.G., Evstrapov A.A. Domestic devices for molecular genetic analysis: developments of the IAP RAS and SINTOL LLC. Russ Mil Med Acad Rep. 2021;40(3): 69-74. doi 10.17816/rmmar76918 (in Russian)Kurochkin V.E., Alekseev Y.I., Petrov D.G., Evstrapov A.A. Domestic devices for molecular genetic analysis: developments of the IAP RAS and SINTOL LLC. Russ Mil Med Acad Rep. 2021;40(3): 69-74. doi 10.17816/rmmar76918 (in Russian)Kutnjak D., Tamisier L., Adams I., Boonham N., Candresse T., Chiumenti M., De Jonghe K., … Rollin J., Rott M., Schumpp O., Massart S., Haegeman A. A primer on the analysis of high-throughput sequencing data for detection of plant viruses. Microorganisms. 2021;9(4):841. doi 10.3390/microorganisms9040841Kutnjak D., Tamisier L., Adams I., Boonham N., Candresse T., Chiumenti M., De Jonghe K., … Rollin J., Rott M., Schumpp O., Massart S., Haegeman A. A primer on the analysis of high-throughput sequencing data for detection of plant viruses. Microorganisms. 2021;9(4):841. doi 10.3390/microorganisms9040841Langmead B., Salzberg S.L. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9(4):357-359. doi 10.1038/nmeth.1923Langmead B., Salzberg S.L. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9(4):357-359. doi 10.1038/nmeth.1923Lebas B., Adams I., Al Rwahnih M., Baeyen S., Bilodeau G.J., Blouin A.G., Boonham N., … Vicente C.S.L., Vossenberg B.T.L.H., Wetzel T., Ziebell H., Massart S. Facilitating the adoption of high-throughput sequencing technologies as a plant pest diagnostic test in laboratories: a step-by-step description. EPPO Bull. 2022;52(2): 394-418. doi 10.1111/epp.12863Lebas B., Adams I., Al Rwahnih M., Baeyen S., Bilodeau G.J., Blouin A.G., Boonham N., … Vicente C.S.L., Vossenberg B.T.L.H., Wetzel T., Ziebell H., Massart S. Facilitating the adoption of high-throughput sequencing technologies as a plant pest diagnostic test in laboratories: a step-by-step description. EPPO Bull. 2022;52(2): 394-418. doi 10.1111/epp.12863Lee H.-K., Kim S.-Y., Yang H.-J., Lee D.-S., Kwon B., Lee D.-Y., Oh J., Lee S.-H. The detection of plant viruses in Korean ginseng (Panax ginseng) through RNA sequencing. Plant Pathol J. 2020;36(6): 643-650. doi 10.5423/PPJ.NT.07.2020.0137Lee H.-K., Kim S.-Y., Yang H.-J., Lee D.-S., Kwon B., Lee D.-Y., Oh J., Lee S.-H. The detection of plant viruses in Korean ginseng (Panax ginseng) through RNA sequencing. Plant Pathol J. 2020;36(6): 643-650. doi 10.5423/PPJ.NT.07.2020.0137Lefeuvre P., Martin D.P., Elena S.F., Shepherd D.N., Roumagnac P., Varsani A. Evolution and ecology of plant viruses. Nat Rev Microbiol. 2019;17(10):632-644. doi 10.1038/s41579-019-0232-3Lefeuvre P., Martin D.P., Elena S.F., Shepherd D.N., Roumagnac P., Varsani A. Evolution and ecology of plant viruses. Nat Rev Microbiol. 2019;17(10):632-644. doi 10.1038/s41579-019-0232-3Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018;34(18):3094-3100. doi 10.1093/bioinformatics/bty191Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018;34(18):3094-3100. doi 10.1093/bioinformatics/bty191Li H., Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics. 2009;25(14):1754-1760. doi 10.1093/bioinformatics/btp324Li H., Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics. 2009;25(14):1754-1760. doi 10.1093/bioinformatics/btp324Liu S., Rodriguez J.S., Munteanu V., Ronkowski C., Sharma N.K., Alser M., Andreace F., … Ganda E., Davenport E.R., Pop M., Koslicki D., Mangul S. Analysis of metagenomic data. Nat Rev Methods Primers. 2025;5(1):5. doi 10.1038/s43586-024-00376-6Liu S., Rodriguez J.S., Munteanu V., Ronkowski C., Sharma N.K., Alser M., Andreace F., … Ganda E., Davenport E.R., Pop M., Koslicki D., Mangul S. Analysis of metagenomic data. Nat Rev Methods Primers. 2025;5(1):5. doi 10.1038/s43586-024-00376-6Luo R., Liu B., Xie Y., Li Z., Huang W., Yuan J., He G., … Li Y., Yang H., Wang J., Lam T.-W., Wang J. SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. GigaScience. 2012;1(1):18. doi 10.1186/2047-217X-1-18Luo R., Liu B., Xie Y., Li Z., Huang W., Yuan J., He G., … Li Y., Yang H., Wang J., Lam T.-W., Wang J. SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. GigaScience. 2012;1(1):18. doi 10.1186/2047-217X-1-18Maina S., Donovan N.J., Plett K., Bogema D., Rodoni B.C. Highthroughput sequencing for plant virology diagnostics and its potential in plant health certification. Front Hortic. 2024;3:1388028. doi 10.3389/fhort.2024.1388028Maina S., Donovan N.J., Plett K., Bogema D., Rodoni B.C. Highthroughput sequencing for plant virology diagnostics and its potential in plant health certification. Front Hortic. 2024;3:1388028. doi 10.3389/fhort.2024.1388028Maliogka V.I., Minafra A., Saldarelli P., Ruiz-García A.B., Glasa M., Katis N., Olmos A. Recent advances on detection and characterization of fruit tree viruses using high-throughput sequencing technologies. Viruses. 2018;10(8):436. doi 10.3390/v10080436Maliogka V.I., Minafra A., Saldarelli P., Ruiz-García A.B., Glasa M., Katis N., Olmos A. Recent advances on detection and characterization of fruit tree viruses using high-throughput sequencing technologies. Viruses. 2018;10(8):436. doi 10.3390/v10080436Massart S., Olmos A., Jijakli H., Candresse T. Current impact and future directions of high throughput sequencing in plant virus diagnostics. Virus Res. 2014;188:90-96. doi 10.1016/j.virusres.2014.03.029Massart S., Olmos A., Jijakli H., Candresse T. Current impact and future directions of high throughput sequencing in plant virus diagnostics. Virus Res. 2014;188:90-96. doi 10.1016/j.virusres.2014.03.029Massart S., Adams I., Al Rwahnih M., Baeyen S., Bilodeau G.J., Blouin A.G., Boonham N., … van de Vossenberg B.T.L.H., Westenberg M., Wetzel T., Ziebell H., Lebas B.S.M. Guidelines for the reliable use of high throughput sequencing technologies to detect plant pathogens and pests. Peer Commun J. 2022;2:e62. doi 10.24072/pcjournal.181Massart S., Adams I., Al Rwahnih M., Baeyen S., Bilodeau G.J., Blouin A.G., Boonham N., … van de Vossenberg B.T.L.H., Westenberg M., Wetzel T., Ziebell H., Lebas B.S.M. Guidelines for the reliable use of high throughput sequencing technologies to detect plant pathogens and pests. Peer Commun J. 2022;2:e62. doi 10.24072/pcjournal.181Menzel P., Ng K.L., Krogh A. Fast and sensitive taxonomic classification for metagenomics with Kaiju. Nat Commun. 2016;7(1):11257. doi 10.1038/ncomms11257Menzel P., Ng K.L., Krogh A. Fast and sensitive taxonomic classification for metagenomics with Kaiju. Nat Commun. 2016;7(1):11257. doi 10.1038/ncomms11257Moubset O., François S., Maclot F., Palanga E., Julian C., Claude L., Fernandez E., … Massart S., Ogliastro M., Martin D.P., Filloux D., Roumagnac P. Virion-associated nucleic acid-based metagenomics: a decade of advances in molecular characterization of plant viruses. Phytopathology. 2022;112(11):2253-2272. doi 10.1094/PHYTO-0322-0096-RVWMoubset O., François S., Maclot F., Palanga E., Julian C., Claude L., Fernandez E., … Massart S., Ogliastro M., Martin D.P., Filloux D., Roumagnac P. Virion-associated nucleic acid-based metagenomics: a decade of advances in molecular characterization of plant viruses. Phytopathology. 2022;112(11):2253-2272. doi 10.1094/PHYTO-0322-0096-RVWNabeshima T., Abe J. High-throughput sequencing indicates novel Varicosavirus, Emaravirus, and Deltapartitivirus infections in Vitis coignetiae. Viruses. 2021;13(5):827. doi 10.3390/v13050827Nabeshima T., Abe J. High-throughput sequencing indicates novel Varicosavirus, Emaravirus, and Deltapartitivirus infections in Vitis coignetiae. Viruses. 2021;13(5):827. doi 10.3390/v13050827Ounit R., Lonardi S. Higher classification sensitivity of short metagenomic reads with CLARK-S. Bioinformatics. 2016;32(24):3823-3825. doi 10.1093/bioinformatics/btw542Ounit R., Lonardi S. Higher classification sensitivity of short metagenomic reads with CLARK-S. Bioinformatics. 2016;32(24):3823-3825. doi 10.1093/bioinformatics/btw542Panno S., Davino S., Caruso A.G., Bertacca S., Crnogorac A., MandićA., Noris E., Matić S. A review of the most common and economically important diseases that undermine the cultivation of tomato crop in the mediterranean basin. Agronomy. 2021;11(11):2188. doi 10.3390/agronomy11112188Panno S., Davino S., Caruso A.G., Bertacca S., Crnogorac A., MandićA., Noris E., Matić S. A review of the most common and economically important diseases that undermine the cultivation of tomato crop in the mediterranean basin. Agronomy. 2021;11(11):2188. doi 10.3390/agronomy11112188Pappas N., Roux S., Hölzer M., Lamkiewicz K., Mock F., Marz M., Dutilh B.E. Virus bioinformatics. In: Encyclopedia of Virology. Elsevier, 2021;124-132. doi 10.1016/B978-0-12-814515-9.00034-5Pappas N., Roux S., Hölzer M., Lamkiewicz K., Mock F., Marz M., Dutilh B.E. Virus bioinformatics. In: Encyclopedia of Virology. Elsevier, 2021;124-132. doi 10.1016/B978-0-12-814515-9.00034-5Pecman A., Kutnjak D., Gutiérrez-Aguirre I., Adams I., Fox A., Boonham N., Ravnikar M. Next generation sequencing for detection and discovery of plant viruses and viroids: comparison of two approaches. Front Microbiol. 2017;8:1998. doi 10.3389/fmicb.2017.01998Pecman A., Kutnjak D., Gutiérrez-Aguirre I., Adams I., Fox A., Boonham N., Ravnikar M. Next generation sequencing for detection and discovery of plant viruses and viroids: comparison of two approaches. Front Microbiol. 2017;8:1998. doi 10.3389/fmicb.2017.01998Peng Y., Leung H.C.M., Yiu S.M., Chin F.Y.L. IDBA-UD: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth. Bioinformatics. 2012;28(11):1420-1428. doi 10.1093/bioinformatics/bts174Peng Y., Leung H.C.M., Yiu S.M., Chin F.Y.L. IDBA-UD: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth. Bioinformatics. 2012;28(11):1420-1428. doi 10.1093/bioinformatics/bts174Piombo E., Abdelfattah A., Droby S., Wisniewski M., Spadaro D., Schena L. Metagenomics approaches for the detection and surveillance of emerging and recurrent plant pathogens. Microorganisms. 2021;9(1):188. doi 10.3390/microorganisms9010188Piombo E., Abdelfattah A., Droby S., Wisniewski M., Spadaro D., Schena L. Metagenomics approaches for the detection and surveillance of emerging and recurrent plant pathogens. Microorganisms. 2021;9(1):188. doi 10.3390/microorganisms9010188Rang F.J., Kloosterman W.P., de Ridder J. From squiggle to basepair: computational approaches for improving nanopore sequencing read accuracy. Genome Biol. 2018;19(1):90. doi 10.1186/s13059-018-1462-9Rang F.J., Kloosterman W.P., de Ridder J. From squiggle to basepair: computational approaches for improving nanopore sequencing read accuracy. Genome Biol. 2018;19(1):90. doi 10.1186/s13059-018-1462-9Roossinck M.J., Martin D.P., Roumagnac P. Plant virus metagenomics: advances in virus discovery. Phytopathology. 2015;105(6):716-727. doi 10.1094/PHYTO-12-14-0356-RVWRoossinck M.J., Martin D.P., Roumagnac P. Plant virus metagenomics: advances in virus discovery. Phytopathology. 2015;105(6):716-727. doi 10.1094/PHYTO-12-14-0356-RVWRose R., Constantinides B., Tapinos A., Robertson D.L., Prosperi M. Challenges in the analysis of viral metagenomes. Virus Evol. 2016; 2(2):vew022. doi 10.1093/ve/vew022Rose R., Constantinides B., Tapinos A., Robertson D.L., Prosperi M. Challenges in the analysis of viral metagenomes. Virus Evol. 2016; 2(2):vew022. doi 10.1093/ve/vew022Rott M., Xiang Y., Boyes I., Belton M., Saeed H., Kesanakurti P., Hayes S., Lawrence T., Birch C., Bhagwat B., Rast H. Application of next generation sequencing for diagnostic testing of tree fruit viruses and viroids. Plant Dis. 2017;101(8):1489-1499. doi 10.1094/PDIS03-17-0306-RERott M., Xiang Y., Boyes I., Belton M., Saeed H., Kesanakurti P., Hayes S., Lawrence T., Birch C., Bhagwat B., Rast H. Application of next generation sequencing for diagnostic testing of tree fruit viruses and viroids. Plant Dis. 2017;101(8):1489-1499. doi 10.1094/PDIS03-17-0306-RERoy S., Coldren C., Karunamurthy A., Kip N.S., Klee E.W., Lincoln S.E., Leon A., Pullambhatla M., Temple-Smolkin R.L., Voelkerding K.V., Wang C., Carter A.B. Standards and guidelines for validating next-generation sequencing bioinformatics pipelines. J Mol Diagn. 2018;20(1):4-27. doi 10.1016/j.jmoldx.2017.11.003Roy S., Coldren C., Karunamurthy A., Kip N.S., Klee E.W., Lincoln S.E., Leon A., Pullambhatla M., Temple-Smolkin R.L., Voelkerding K.V., Wang C., Carter A.B. Standards and guidelines for validating next-generation sequencing bioinformatics pipelines. J Mol Diagn. 2018;20(1):4-27. doi 10.1016/j.jmoldx.2017.11.003Rubino L., Abrahamian P., An W., Aranda M.A., Ascencio-Ibañez J.T., Bejerman N., Blouin A.G., … Whitfield A.E., Wylie S.J., Yang C., Zerbini F.M., Zhang S. Summary of taxonomy changes ratified by the International Committee on Taxonomy of Viruses from the Plant Viruses Subcommittee, 2025. J Gen Virol. 2025;106(7):002114. doi 10.1099/jgv.0.002114Rubino L., Abrahamian P., An W., Aranda M.A., Ascencio-Ibañez J.T., Bejerman N., Blouin A.G., … Whitfield A.E., Wylie S.J., Yang C., Zerbini F.M., Zhang S. Summary of taxonomy changes ratified by the International Committee on Taxonomy of Viruses from the Plant Viruses Subcommittee, 2025. J Gen Virol. 2025;106(7):002114. doi 10.1099/jgv.0.002114Sayers E.W., Beck J., Bolton E.E., Brister J.R., Chan J., Connor R., Feldgarden M., … Wang J., Ye J., Zellers E., Schneider V.A., Pruitt K.D. Database resources of the National Center for Biotechnology Information in 2025. Nucleic Acids Res. 2025;53(D1):D20-D29. doi 10.1093/nar/gkae979Sayers E.W., Beck J., Bolton E.E., Brister J.R., Chan J., Connor R., Feldgarden M., … Wang J., Ye J., Zellers E., Schneider V.A., Pruitt K.D. Database resources of the National Center for Biotechnology Information in 2025. Nucleic Acids Res. 2025;53(D1):D20-D29. doi 10.1093/nar/gkae979Shvets D., Sandomirsky K., Porotikova E., Vinogradova S. Metagenomic analysis of ampelographic collections of Dagestan revealed the presence of two novel grapevine viruses. Viruses. 2022;14(12): 2623. doi 10.3390/v14122623Shvets D., Sandomirsky K., Porotikova E., Vinogradova S. Metagenomic analysis of ampelographic collections of Dagestan revealed the presence of two novel grapevine viruses. Viruses. 2022;14(12): 2623. doi 10.3390/v14122623Simpson J.T., Wong K., Jackman S.D., Schein J.E., Jones S.J.M., Birol İ. ABySS: a parallel assembler for short read sequence data. Genome Res. 2009;19(6):1117-1123. doi 10.1101/gr.089532.108Simpson J.T., Wong K., Jackman S.D., Schein J.E., Jones S.J.M., Birol İ. ABySS: a parallel assembler for short read sequence data. Genome Res. 2009;19(6):1117-1123. doi 10.1101/gr.089532.108Sović I., Šikić M., Wilm A., Fenlon S.N., Chen S., Nagarajan N. Fast and sensitive mapping of nanopore sequencing reads with GraphMap. Nat Commun. 2016;7(1):11307. doi 10.1038/ncomms11307Sović I., Šikić M., Wilm A., Fenlon S.N., Chen S., Nagarajan N. Fast and sensitive mapping of nanopore sequencing reads with GraphMap. Nat Commun. 2016;7(1):11307. doi 10.1038/ncomms11307Sun K., Liu Y., Zhou X., Yin C., Zhang P., Yang Q., Mao L., Shentu X., Yu X. Nanopore sequencing technology and its application in plant virus diagnostics. Front Microbiol. 2022;13:939666. doi 10.3389/fmicb.2022.939666Sun K., Liu Y., Zhou X., Yin C., Zhang P., Yang Q., Mao L., Shentu X., Yu X. Nanopore sequencing technology and its application in plant virus diagnostics. Front Microbiol. 2022;13:939666. doi 10.3389/fmicb.2022.939666Sutton T.D.S., Clooney A.G., Ryan F.J., Ross R.P., Hill C. Choice of assembly software has a critical impact on virome characterisation. Microbiome. 2019;7(1):12. doi 10.1186/s40168-019-0626-5Sutton T.D.S., Clooney A.G., Ryan F.J., Ross R.P., Hill C. Choice of assembly software has a critical impact on virome characterisation. Microbiome. 2019;7(1):12. doi 10.1186/s40168-019-0626-5Turco S., Golyaev V., Seguin J., Gilli C., Farinelli L., Boller T., Schumpp O., Pooggin M.M. Small RNA-omics for virome reconstruction and antiviral defense characterization in mixed infections of cultivated Solanum plants. Mol Plant Microbe Interact. 2018; 31(7):707-723. doi 10.1094/MPMI-12-17-0301-RTurco S., Golyaev V., Seguin J., Gilli C., Farinelli L., Boller T., Schumpp O., Pooggin M.M. Small RNA-omics for virome reconstruction and antiviral defense characterization in mixed infections of cultivated Solanum plants. Mol Plant Microbe Interact. 2018; 31(7):707-723. doi 10.1094/MPMI-12-17-0301-RVanderWeele T.J., Ding P. Sensitivity analysis in observational research: introducing the E-value. Ann Intern Med. 2017;167(4):268-274. doi 10.7326/M16-2607VanderWeele T.J., Ding P. Sensitivity analysis in observational research: introducing the E-value. Ann Intern Med. 2017;167(4):268-274. doi 10.7326/M16-2607Villamor D.E.V., Ho T., Al Rwahnih M., Martin R.R., Tzanetakis I.E. High throughput sequencing for plant virus detection and discovery. Phytopathology. 2019;109(5):716-725. doi 10.1094/PHYTO-07-180257-RVWVillamor D.E.V., Ho T., Al Rwahnih M., Martin R.R., Tzanetakis I.E. High throughput sequencing for plant virus detection and discovery. Phytopathology. 2019;109(5):716-725. doi 10.1094/PHYTO-07-180257-RVWVinogradova S., Porotikova E., Navrotskaya E., Galbacs Z.N., Massart S., Varallyay E. The first virome of a Russian vineyard. Plants. 2023;12(18):3292. doi 10.3390/plants12183292Vinogradova S., Porotikova E., Navrotskaya E., Galbacs Z.N., Massart S., Varallyay E. The first virome of a Russian vineyard. Plants. 2023;12(18):3292. doi 10.3390/plants12183292Vivek A.T., Zahra S., Kumar S. From current knowledge to best practice: a primer on viral diagnostics using deep sequencing of virusderived small interfering RNAs (vsiRNAs) in infected plants. Methods. 2020;183:30-37. doi 10.1016/j.ymeth.2019.10.009Vivek A.T., Zahra S., Kumar S. From current knowledge to best practice: a primer on viral diagnostics using deep sequencing of virusderived small interfering RNAs (vsiRNAs) in infected plants. Methods. 2020;183:30-37. doi 10.1016/j.ymeth.2019.10.009Wang Z., Qin L., Liu J., Jiang L., Zou X., Chen X., Song F., Dai H., Hou Y. Forensic nanopore sequencing of microhaplotype markers using QitanTech’s QNome. Forensic Sci Int Genet. 2022;57:102657. doi 10.1016/j.fsigen.2021.102657Wang Z., Qin L., Liu J., Jiang L., Zou X., Chen X., Song F., Dai H., Hou Y. Forensic nanopore sequencing of microhaplotype markers using QitanTech’s QNome. Forensic Sci Int Genet. 2022;57:102657. doi 10.1016/j.fsigen.2021.102657White D.J., Wang J., Hall R.J. Assessing the impact of assemblers on virus detection in a de novo metagenomic analysis pipeline. J Comput Biol. 2017;24(9):874-881. doi 10.1089/cmb.2017.0008White D.J., Wang J., Hall R.J. Assessing the impact of assemblers on virus detection in a de novo metagenomic analysis pipeline. J Comput Biol. 2017;24(9):874-881. doi 10.1089/cmb.2017.0008Wick R.R., Judd L.M., Gorrie C.L., Holt K.E. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol. 2017;13(6):e1005595. doi 10.1371/journal.pcbi.1005595Wick R.R., Judd L.M., Gorrie C.L., Holt K.E. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol. 2017;13(6):e1005595. doi 10.1371/journal.pcbi.1005595Wood D.E., Lu J., Langmead B. Improved metagenomic analysis with Kraken 2. Genome Biol. 2019;20(1):257. doi 10.1186/s13059-019-1891-0Wood D.E., Lu J., Langmead B. Improved metagenomic analysis with Kraken 2. Genome Biol. 2019;20(1):257. doi 10.1186/s13059-019-1891-0Zerbino D.R., Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008;18(5):821-829. doi 10.1101/gr.074492.107Zerbino D.R., Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008;18(5):821-829. doi 10.1101/gr.074492.107Zheng Y., Gao S., Padmanabhan C., Li R., Galvez M., Gutierrez D., Fuentes S., Ling K.-S., Kreuze J., Fei Z. VirusDetect: an automated pipeline for efficient virus discovery using deep sequencing of small RNAs. Virology. 2017;500:130-138. doi 10.1016/j.virol.2016.10.017Zheng Y., Gao S., Padmanabhan C., Li R., Galvez M., Gutierrez D., Fuentes S., Ling K.-S., Kreuze J., Fei Z. VirusDetect: an automated pipeline for efficient virus discovery using deep sequencing of small RNAs. Virology. 2017;500:130-138. doi 10.1016/j.virol.2016.10.017Zhuravlyov V.S., Dolgikh V.V., Timofeev S.A., Gannibal F.B. RNA interference method in plant protection against insect pests. Plant Prot News. 2022;105(1):28-39. doi 10.31993/2308-6459-2022-105-1-15219Zhuravlyov V.S., Dolgikh V.V., Timofeev S.A., Gannibal F.B. RNA interference method in plant protection against insect pests. Plant Prot News. 2022;105(1):28-39. doi 10.31993/2308-6459-2022-105-1-15219Zubov V.V., Chemeris D.A., Vasilov R.G., Kurochkin V.E., Alekseev Ya.I. Brief history of high-throughput nucleic acid sequencing methods. Biomics. 2021;13(1):27-46. doi 10.31301/2221-6197.bmcs.2021-4Zubov V.V., Chemeris D.A., Vasilov R.G., Kurochkin V.E., Alekseev Ya.I. Brief history of high-throughput nucleic acid sequencing methods. Biomics. 2021;13(1):27-46. doi 10.31301/2221-6197.bmcs.2021-4

The authors declare that there are no conflicts of interest present.