共价闭合的环状RNA与真核长非编码RNA家族是新的,这是由于外显子的5'-3'反向剪接而出现的,内含子,或跨越亲本基因的基因间区域。由于其独特的结构和稳定性,环状RNA具有多种功能特性,如微小RNA和蛋白质海绵,基因表达的直接和间接调节剂,蛋白质翻译,和许多未经证实的活动,除了潜在的生物标志物。然而,由于它们的丰度低,大多数全球环状RNA鉴定是通过基于高通量NGS的方法进行的,需要数百万个测序读数。方法论进步的这种滞后要求更新,更精致,和高效的识别技术。这里,我们的目标是显示我们以前报道的模板依赖性多重置换扩增(tdMDA)-NGS方法的改进版本,通过叠加核糖体耗尽步骤和使用H减去逆转录酶和RNaseH。在最小化线性和核糖体RNA含量后,使用高度复制的Phi29DNA聚合酶的tdMDA的含义进一步增强了其对低NGS深度的深渊环状表达RNA的检测极限,从而降低了鉴定单个环状RNA的成本。与使用DCC和CIRI2管道的传统circRNA测序方法相比,通过改进的tdMDA-NGS方法鉴定出总环状RNA的>11倍和>6倍增加,分别,来自水稻亚种。Indica.此外,在HeLa细胞系中也断言了改进的tdMDA-NGS方法的可靠性,与现有的传统circRNA测序方法相比,显示出显着的倍数差异。在确定的环状RNA中,一个显著的百分比从水稻(~58%)和HeLa细胞系(~84%)被发现与以前报道的环状RNA相匹配,这表明改进的tdMDA-NGS方法可以适应于检测和表征来自不同生物系统的环状RNA。
Covalently closed circular RNAs are neoteric to the eukaryotic family of long non-coding RNAs emerging as a result of 5\'-3\' backsplicing from exonic, intronic, or intergenic regions spanning the parental gene. Owing to their unique structure and stability, circular RNAs have a multitude of functional properties such as micro-RNA and protein sponges, direct and indirect modulators of gene expression, protein translation, and many unproven activities apart from being potential biomarkers. However, due to their low abundance, most of the global circular RNA identification is carried out by high-throughput NGS-based approaches requiring millions of sequencing reads. This lag in methodological advancements demands for newer, more refined, and efficient identification techniques. Here, we aim to show an improved version of our previously reported template-dependent multiple displacement amplification (tdMDA)-NGS method by superimposing the ribosomal depletion step and use of H minus reverse transcriptase and RNase H. Implication of tdMDA using highly replicative Phi29 DNA polymerase after minimizing the linear and ribosomal RNA content further intensifies its detection limit toward even the abysmally expressing circular RNA at a low NGS depth, thereby decreasing the cost of identifying a single circular RNA. A >11-fold and >6-fold increase in total circular RNA was identified from the improved-tdMDA-NGS method over the traditional method of circRNA sequencing using DCC and CIRI2 pipelines, respectively, from Oryza sativa subsp. Indica. Furthermore, the reliability of the improved-tdMDA-NGS method was also asserted in HeLa cell lines, showing a significant fold difference in comparison with the existing traditional method of circRNA sequencing. Among the identified circular RNAs, a significant percentage from both rice (∼58%) and HeLa cell lines (∼84%) is found to be matched with the previously reported circular RNAs, suggesting that the improved-tdMDA-NGS method can be adapted to detect and characterize the circular RNAs from different biological systems.