Plant virome analysis by high-throughput sequencing: concepts and approaches

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.

1. Atallah 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.2011.11055

2. Baizan-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.0.001210

3. Bankevich 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.2012.0021

4. Belkina 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/v15122429

5. Bolger A.M., Lohse M., Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114-2120. doi 10.1093/bioinformatics/btu170

6. Buchfink B., Xie C., Huson D.H. Fast and sensitive protein alignment using DIAMOND. Nat Methods. 2015;12(1):59-60. doi 10.1038/nmeth.3176

7. Camacho 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 10.1186/1471-2105-10-421

8. Chevreux 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;45-56

9. Chin 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.4035

10. Cholet 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 10.1111/1462-2920.15017

11. Cobbin 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.2021.09.007

12. Crossley 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 10.1177/1040638720905833

13. Dobin 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.bi1114s51

14. Fall 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 10.1111/1755-0998.14111

15. Filloux 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_18

16. Fitzpatrick 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.2021.621719

17. Gaafar 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.0237951

18. Gallo 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.2020.101570

19. Han 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/microorganisms12122482

20. Hu 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/s12859-023-05589-2

21. Javaran 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/plants10112355

22. Jones R.A.C. Global plant virus disease pandemics and epidemics. Plants. 2021;10(2):233. doi 10.3390/plants10020233

23. Kim 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.210641.116

24. Kim 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/s41587-019-0201-4

25. Koren 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.215087.116

26. 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)

27. 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/microorganisms9040841

28. Langmead B., Salzberg S.L. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9(4):357-359. doi 10.1038/nmeth.1923

29. Lebas 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.12863

30. Lee 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.07.2020.0137

31. Lefeuvre 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/s41579-019-0232-3

32. Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018;34(18):3094-3100. doi 10.1093/bioinformatics/bty191

33. Li H., Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics. 2009;25(14):1754-1760. doi 10.1093/bioinformatics/btp324

34. Liu 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/s43586-024-00376-6

35. Luo 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-1-18

36. Maina 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.2024.1388028

37. Maliogka 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/v10080436

38. Massart 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.2014.03.029

39. Massart 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.181

40. Menzel P., Ng K.L., Krogh A. Fast and sensitive taxonomic classification for metagenomics with Kaiju. Nat Commun. 2016;7(1):11257. doi 10.1038/ncomms11257

41. Moubset 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-RVW

42. Nabeshima T., Abe J. High-throughput sequencing indicates novel Varicosavirus, Emaravirus, and Deltapartitivirus infections in Vitis coignetiae. Viruses. 2021;13(5):827. doi 10.3390/v13050827

43. Ounit R., Lonardi S. Higher classification sensitivity of short metagenomic reads with CLARK-S. Bioinformatics. 2016;32(24):3823-3825. doi 10.1093/bioinformatics/btw542

44. Panno 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/agronomy11112188

45. Pappas 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/B978-0-12-814515-9.00034-5

46. Pecman 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.2017.01998

47. Peng 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/bts174

48. Piombo 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/microorganisms9010188

49. Rang 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/s13059-018-1462-9

50. Roossinck 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-RVW

51. Rose 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/vew022

52. Rott 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-RE

53. Roy 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.2017.11.003

54. Rubino 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.0.002114

55. Sayers 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/gkae979

56. Shvets 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/v14122623

57. Simpson 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.089532.108

58. Sović 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/ncomms11307

59. Sun 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.2022.939666

60. Sutton 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/s40168-019-0626-5

61. Turco 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-R

62. VanderWeele T.J., Ding P. Sensitivity analysis in observational research: introducing the E-value. Ann Intern Med. 2017;167(4):268-274. doi 10.7326/M16-2607

63. Villamor 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-RVW

64. Vinogradova 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/plants12183292

65. Vivek 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.2019.10.009

66. Wang 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.2021.102657

67. White 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.2017.0008

68. Wick 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.1005595

69. Wood D.E., Lu J., Langmead B. Improved metagenomic analysis with Kraken 2. Genome Biol. 2019;20(1):257. doi 10.1186/s13059-019-1891-0

70. Zerbino 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.074492.107

71. Zheng 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.2016.10.017

72. Zhuravlyov 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 10.31993/2308-6459-2022-105-1-15219

73. Zubov 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