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Series GSE17164 Query DataSets for GSE17164
Status Public on Apr 12, 2010
Title Structural and Functional Analysis of Viral siRNAs using 454 sequencing
Organisms Nicotiana benthamiana; Cymbidium ringspot virus
Experiment type Non-coding RNA profiling by high throughput sequencing
Summary We profiled vsiRNAs using two different high-throughput sequencing platforms and also developed a hybridisation-based array approach. The profiles obtained through the Solexa platform and by hybridisation were very similar to each other but different from the 454 profile. Both deep sequencing techniques revealed a strong bias in vsiRNAs for the positive strand of the virus and identified regions on the viral genome that produced vsiRNA in much higher abundance than other regions. The hybridisation approach also showed that the position of highly abundant vsiRNAs were the same in different plant species and in the absence of RDR6. We used the TerminatorTM 5'-Phosphate-Dependent Exonuclease to study the 5' end of vsiRNAs and showed that a perfect control duplex was not digested by the enzyme without denaturation and that the efficiency of the Terminator was strongly affected by the concentration of the substrate. We found that most vsiRNAs have 5' monophosphates, which was also confirmed by profiling short RNA libraries following either direct ligation of adapters to the 5' end of short RNAs or after replacing any potential 5' ends with monophosphates. The Terminator experiments also showed that vsiRNAs were not perfect duplexes. Using a sensor construct we also found that regions from the viral genome that were not complementary to highly abundant vsiRNAs were targeted in planta just as efficiently as regions recognised by abundant vsiRNAs.

Different high-throughput sequencing techniques have different reproducible sequence bias and generate different profiles of short RNAs. The Terminator exonuclease does not process double-stranded RNA and because short RNAs can quickly re-anneal at high concentration, this assay can be misleading if the substrate is not denatured and not analysed in a dilution series. The sequence profiles and Terminator digests suggest that CymRSV siRNAs are produced from the structured positive strand rather than from perfect double-stranded RNA or by RNA-dependent RNA polymerase.
Overall design Size-fractionated (19-24 nt) small RNA from early systemic leaves of CymRSV-infected Nicotiana benthamiana total RNA extracts was ligated to adapters, purified again and reverse transcribed. After PCR amplification, the sample was subjected to 454 high throughput pyrosequencing. Please see for details of the sequencing technology.
Contributor(s) Szittya G, Moxon S, Pantaleo V, Toth G, Rusholme-Pilcher R, Burgyan J, Dalmay T
Citation(s) 20368973
Submission date Jul 17, 2009
Last update date Jun 11, 2013
Contact name Simon Moxon
Organization name University of East Anglia
Street address University of East Anglia
City Norwich
State/province Norfolk
ZIP/Postal code NR4 7TJ
Country United Kingdom
Platforms (1)
GPL15026 454 GS (Cymbidium ringspot virus; Nicotiana benthamiana)
Samples (2)
GSM429419 454-sequenced Cymbidium Ringspot Virus (CymRSV)-derived siRNAs
GSM429420 454-sequenced P19 silencing suppressor mutant Cymbidium Ringspot Virus (Cym19stop)-derived siRNAs
This SubSeries is part of SuperSeries:
GSE17278 Structural and Functional Analysis of Viral siRNAs
BioProject PRJNA123661

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE17164_RAW.tar 13.9 Mb (http)(custom) TAR (of FNA, QUAL)
GSE17164_sequence_annotations.txt.gz 469.5 Kb (ftp)(http) TXT
Raw data provided as supplementary file
Processed data included within Sample table

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