Abstract
Small RNAs (sRNAs) have emerged as one of the most important regulators of gene expression in eukaryotes. sRNAs are intermediate molecules as well as end products in the antiviral defense pathway called RNA interference in plants and animals. Profiling of sRNAs using next-generation sequencing technologies has identified a number of plant viruses that have never been reported previously, and has provided a deeper view of virus populations in a plant that cannot be achieved by conventional methods like PCR and ELISA. In this chapter, we describe the methodology of deep sequencing of sRNAs. The high-throughput and highly sensitive method will revolutionize the identification of plant viruses and the study of molecular plant–virus interactions.
Key words
- RNAi
- Small RNAs
- Deep sequencing
- Virus identification
- Bioinformatics
This is a preview of subscription content, access via your institution.
Buying options
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Jones R (2006) RNA silencing sheds light on the RNA world. PLoS Biol 4:e448
Vasudevan S, Tong Y, Steitz JA (2007) Switching from repression to activation: microRNAs can upregulate translation. Science 318:1931–1934
Wang XB, Jovel J, Udomporn P, Wang X, Wu Q, Li WX, Gasciolli V, Vaucheret H, Ding SW (2011) The 21-nucleotide, but not 22-nucleotide, viral secondary small interfering RNAs direct potent antiviral defense by two cooperative argonautes in Arabidopsis thaliana. Plant Cell 23:1625–1638
Almeida R, Allshire RC (2005) RNA silencing and genome regulation. Trends Cell Biol 15:251–258
Tomari Y, Zamore PD (2005) Perspective: machines for RNAi. Genes Dev 19:517–529
Chapman EJ, Carrington JC (2007) Specialization and evolution of endogenous small RNA pathways. Nat Rev Genet 8:884–896
Chen X (2010) Small RNAs―secrets and surprises of the genome. Plant J 61:941–958
Llave C, Kasschau KD, Rector MA, Carrington JC (2002) Endogenous and silencing associated small RNAs in plants. Plant Cell 14:1605–1619
Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP (2002) MicroRNAs in plants. Genes Dev 16:1616–1626
Sunkar R, Zhu JK (2004) Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16:2001–2019
Szittya G, Moxon S, Santos DM, Jing R, Fevereiro MP, Moulton V, Dalmay T (2008) High throughput sequencing of Medicago truncatula short RNAs identifies eight new miRNA families. BMC Genomics 9:593
Zhao CZ, Xia H, Frazier TP, Yao YY, Bi YP, Li AQ, Li MJ, Li CS, Zhang BH, Wang XJ (2010) Deep sequencing identifies novel and conserved microRNAs in peanuts (Arachis hypogaea L.). BMC Plant Biol 10:3
Ding SW, Voinnet O (2007) Antiviral immunity directed by small RNAs. Cell 130:413–426
Mlotshwa S, Pruss GJ, Vance V (2008) Small RNAs in viral infection and host defense. Trends Plant Sci 13:375–382
Donaire L, Wany Y, Gonzalez-Ibeas D, Mayer KF, Aranda MA, Llave C (2009) Deep sequencing of plant viral small RNAs reveals effective and widespread targeting of viral genomes. Virology 392:203–214
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 diagnosis, discovery and sequencing of viruses. Virology 388:1–7
Rwahnih MA, 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
Carra A, Mica E, Gambino G, Pindo M, Moser C, Pè ME, Schubert A (2009) Cloning and characterization of small non-coding RNAs from grape. Plant J 59:750–763
Zhang Y, Singh K, Kaur R, Qiu W (2011) Association of a novel DNA virus with the grapevine vein-clearing and vine decline syndrome. Phytopathology. doi:10.1094/PHYTO-02-11-0034
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor
Ghawana S, Paul A, Kumar H, Kumar A, Singh H, Bhardwaj PK, Rani A, Singh RS, Raizada J, Singh K, Kumar S (2011) An RNA isolation system for plant tissues rich in secondary metabolites. BMC Res Notes 4:85
Hafner M, Landgraf P, Ludwig J, Rice A, Ojo T, Lin C, Tuschl T (2008) Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing. Methods 44:3–12
Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
Warren RL, Sutton GG, Jones SJM, Holt RA (2007) Assembling millions of short DNA sequences using SSAKE. Bioinformatics 23:500–501
Zerbino DR, Birney E (2008) Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18:821–829
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Singh, K., Kaur, R., Qiu, W. (2012). New Virus Discovery by Deep Sequencing of Small RNAs. In: Jin, H., Gassmann, W. (eds) RNA Abundance Analysis. Methods in Molecular Biology, vol 883. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-839-9_14
Download citation
DOI: https://doi.org/10.1007/978-1-61779-839-9_14
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-838-2
Online ISBN: 978-1-61779-839-9
eBook Packages: Springer Protocols