Abstract
Next-generation sequencing (NGS) technologies, for the first time, provide a truly “complete” representation of the viral (and other) pathogens present in a host organism. This is achieved in an unbiased way, and without any prior biological or molecular knowledge of these pathogen(s). During recent years a number of broad approaches, for most of the popular NGS platforms, have been developed. Here we describe such a protocol—one that accurately and reliably analyze viruses (and viroids) infecting grapevine. Our strategy relies on the synthesis of cDNA sequencing libraries from dsRNA, extracted from diseased grapevine tissues; the sequencing of these on an Illumina platform, and a streamlined bioinformatics pipeline to analyze the NGS data, yielding the virus composition (virome) of a specific grapevine tissue type, organ, entire plant, or even a vineyard.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Martelli G (2014) Directory of virus and virus-like diseases of the grapevine and their agents. J Plant Pathol 96(1S):7–8
Nimmo-Bell (2006) The economic effects and financial impact of GLRaV3. Nimmo-Bell, Hastings, pp 1–18, A Nimmo-Bell 379 Publication
Freeborough M-J, Burger JT (2008) Leafroll: economic implications. Wynboer. http://www.wynboer.co.za/recentarticles/200812-leafroll.php3. Accessed 30 Sept 2014
Atallah SS, Gómez MI, Fuchs MF, Martinson TE (2012) Economic impact of Grapevine leafroll disease on Vitis vinifera cv. Cabernet franc in Finger Lakes vineyards of New York. Am J Enol Viticult 63:73–79
Maree HJ, Almeida RP, Bester R, Chooi KM, Cohen D, Dolja VV, Fuchs MF, Golino DA, Jooste AE, Martelli GP, Naidu RA, Rowhani A, Saldarelli P, Burger JT (2013) Grapevine leafroll-associated virus 3. Front Microbiol 4:82
Clark MF, Adams AN (1977) Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J Gen Virol 34:475–483
Al Rwahnih M, Daubert S, Golino D, Rowhani A (2009) Deep sequencing analysis of RNAs from a grapevine showing Syrah decline symptoms reveals a multiple virus infection that includes a novel virus. Virology 387:395–401
Kreuze JF, Perez A, Untiveros M, Quispe D, Fuentes S, Barker I, Simon R (2009) Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: a generic method for diagnosis, discovery and sequencing of viruses. Virology 388:1–7
Coetzee B, Freeborough MJ, Maree HJ, Celton JM, Rees DJ, Burger JT (2010) Deep sequencing analysis of viruses infecting grapevines: virome of a vineyard. Virology 400:157–163
Alabi OJ, Zheng Y, Jagadeeswaran G, Sunkar R, Naidu RA (2012) High-throughput sequence analysis of small RNAs in grapevine (Vitis vinifera L.) affected by grapevine leafroll disease. Mol Plant Pathol 13:1060–1076
Boonham N, Kreuze J, Winterc S, Van der Vlugt R, Bergervoet J, Tomlinson J, Mumford R (2014) Methods in virus diagnostics: from ELISA to next generation sequencing. Virus Res 186:20–31
Massart S, Olmos A, Candresse T (2014) Current impact and future directions of high throughput sequencing in plant virus diagnostics. Virus Res 188:90–96
Buermans HP, Den Dunnen JT (2014) Next generation sequencing technology: advances and applications. Biochim Biophys Acta 1842:1932–1941
Van Dijk EL, Auger H, Jaszczyszyn Y, Thermes C (2014) Ten years of next-generation sequencing technology. Trends Genet 30:418–426
Morris TJ, Dodds JA (1979) Isolation and analysis of double-stranded RNA from virus-infected plant and fungal tissue. Phytopathology 69:854–858
Acknowledgements
We hereby acknowledge financial support from Winetech and the THRIP programme of the National Research Foundation. We thank Beatrix Coetzee for critical reading of the manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Burger, J.T., Maree, H.J. (2015). Metagenomic Next-Generation Sequencing of Viruses Infecting Grapevines. In: Lacomme, C. (eds) Plant Pathology. Methods in Molecular Biology, vol 1302. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2620-6_23
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2620-6_23
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2619-0
Online ISBN: 978-1-4939-2620-6
eBook Packages: Springer Protocols