Computational protocol: Discovery of Replicating Circular RNAs by RNA-Seq and Computational Algorithms

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Protocol publication

[…] The small RNA libraries were constructed using Illumina's small RNA sample preparation Kit (Invitrogen, USA) following Illumina's method. The protocols for sRNA purification, adaptor ligation, RT-PCR amplification, library purification and high-throughput DNA sequencing on an Illumina HiSeq-2000 have been reported previously , . Two sRNA libraries of an old grapevine plant and an apple tree were sequenced. Raw data from the Illumina platform were first processed to trim adaptor and barcode sequences. Reads of 18-30 nt were extracted from the obtained trimmed reads to generate sRNA libraries for assembly. The sRNA library of a peach tree infected with PLMVd (accession no. GSM465746) and the small RNA library from a grapevine tree cultivar Pinot noir ENTAV115 (accession no. GSE18405) were downloaded from the NCBI Gene Expression Omnibus (GEO) database. All of the prepared sRNA libraries were fed into an in-house pipeline. Briefly, exogenous sRNA was enriched by subtracting sRNA derived from the host genome using the Bowtie2 with default parameters . The highly enriched exogenous siRNA from each sample were assembled de novo using Velvet and PFOR /PFOR2. The resulting contigs were queried against the GenBank nt and nr databases using the BLAST program .The RNA-seq libraries of potato samples were constructed with a modified Not Not So Random (NNSR) sequencing method . Two libraries of potato were sequenced using an Ion-torrent sequencer according to the manufacturer's instructions. [...] The secondary structures with minimum free energy for GLVd and AHVd-like RNA were predicted by the circular version of the MFold program . The obtained secondary structures were further edited for print by RnaViz 2 . To search for possible kissing-loops in AHVd-like RNA, the Kinefold web server was used with default parameters. […]

Pipeline specifications

Software tools Mfold, RnaViz, Kinefold
Application RNA structure analysis
Organisms Malus domestica
Diseases Infection