Assessment of different enrichment methods revealed the optimal approach to identify bovine circRnas.

Although circular RNAs (circRNAs) play important roles in regulating gene expression, the understanding of circRNAs in livestock animals is scarce due to the significant challenge to characterize them from a biological sample. In this study, we assessed the outcomes of bovine circRNA identification using six enrichment approaches with the combination of ribosomal RNAs removal (); linear RNAs degradation (); linear RNAs and RNAs with structured 3' ends degradation (); ribosomal RNAs coupled with linear RNAs elimination (); ribosomal RNA, linear RNAs and RNAs with poly (A) tailing elimination (); and ribosomal RNA, linear RNAs and RNAs with structured 3' ends elimination (), respectively. RNA-sequencing analysis revealed that different approaches led to varied ratio of uniquely mapped reads, false-positive rate of identifying circRNAs, and the number of circRNAs per million clean reads ( <0.05). Out of 2,285 and 2,939 highly confident circRNAs identified in liver and rumen tissues, respectively, 308 and 260 were commonly identified from five methods, with Ribo-RTP method identified the highest number of circRNAs. Besides, 507 of 4,051 identified bovine highly confident circRNAs had shared splicing sites with human circRNAs. The findings from this work provide optimized methods to identify bovine circRNAs from cattle tissues for downstream research of their biological roles in cattle.