NGS Methodologies and Computational Algorithms for the Prediction and Analysis of Plant Circular RNAs

Circular RNAs (circRNAs) are a class of single-stranded RNAs derived from exonic, intronic, and intergenic regions from precursor messenger RNAs (pre-mRNA), where a noncanonical back-splicing event occurs, in which the 5' and 3' ends are attached by covalent bond. CircRNAs participate in the regulation of gene expression at the transcriptional and posttranscriptional level primarily as miRNA and RNA-binding protein (RBP) sponges, but also involved in the regulation of alternative RNA splicing and transcription. CircRNAs are widespread and abundant in plants where they have been involved in stress responses and development. Through the analysis of all publications in this field in the last five years, we can summarize that the identification of these molecules is carried out through next generation sequencing studies, where samples have been previously treated to eliminate DNA, rRNA, and linear RNAs as a means to enrich circRNAs. Once libraries are prepared, they are sequenced and subsequently studied from a bioinformatics point of view. Among the different tools for identifying circRNAs, we can highlight CIRI as the most used (in 60% of the published studies), as well as CIRCExplorer (20%) and find_circ (20%). Although it is recommended to use more than one program in combination, and preferably developed specifically to treat with plant samples, this is not always the case. It should also be noted that after identifying these circular RNAs, most of the authors validate their findings in the laboratory in order to obtain bona fide results.