Computational protocol: Identification of Circular RNAs from the Parental Genes Involved in Multiple Aspects of Cellular Metabolism in Barley

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Protocol publication

[…] The sequencing reads were trimmed for the first 20 and the last 6 nucleotides using Trimmomatic-0.33 (Bolger et al., ). In addition, a quality filtering was applied to both ends of the reads for nucleotides with quality scores lower than 22 as well as sliding trimming with a window size of 4 and an average quality score of 15 in Phred-33 scoring system. Employing a split mapping algorithm, BWA-MEM (bwa-0.7.12; Li, ) was used to map more than 514 million trimmed paired reads (2 × 76 bases) onto the barley genome release 26 available at EnsemblPlants (http://plants.ensembl.org/index.html). To avoid the process of read-trimming during mapping, we applied a large clipping penalty (-L 20000). The SAM file of alignment was inspected by the software CIRI v1.2 (Gao et al., ) to identify the paired reads supporting the junctions in circular RNA. The identified circular RNAs were further filtered out manually. Due to the possibility of contig mis-assembly, we first excluded the circular RNA candidates if the flanking regions of their junctions were mapped onto two different genomic contigs. Second, circular RNAs were not further considered if their junction regions were evidenced, i.e., the occurrence of back-splicing was rejected, by any publicly-available linear transcript or genomic sequence at NCBI. Recalling the functioning mechanisms of circular RNAs (Lasda and Parker, ; Andreeva and Cooper, ), repeated nucleotide motives can be of the utmost importance. We, therefore, inspected the identified circular RNAs for a maximum of two different nucleotide patterns with highest scores. We used the CLC software which creates a new Hidden Markov Model based on the selected sequence to find repeated nucleotide motives. We adjusted the pattern length varying from 4 to 25 bases. […]

Pipeline specifications

Software tools Trimmomatic, BWA, CIRI
Application RNA-seq analysis
Organisms Hordeum vulgare
Chemicals Amino Acids, Iron, Zinc