Circular RNA Identification and Characterization with CircRNAFlow: A Bioinformatics Approach

Circular RNAs (circRNAs) play various roles in gene regulation and are known to be associated with the development of disease. They can act as microRNA sponges, interact with RNA-binding proteins, and even contain open reading frames (ORFs) that are translated to generate proteins. Scientists hope to better understand these covalently closed ring-like structures as they are not well-characterized.Here, we present CircRNAFlow, a free and open-source Nextflow-based pipeline for in silico circRNA identification and characterization. This next-generation sequencing (NGS) pipeline takes paired-end FASTQ files as input and produces tables and graphical images that report identified circRNAs and their corresponding characterizations. Reports include CircTest to compare circRNA expression to host gene expression and across groups. Characterization includes ClusterProfiler, CRAFT (CircRNA Function prediction Tool), and deepTarget, which explore the regulatory roles of identified circRNAs.In this chapter, we discuss the bioinformatics analyses carried out by the incorporated third-party software of CircRNAFlow [namely, FastQC, Flexbar, Bowtie2, STAR (Spliced Transcripts Alignment to a Reference), DCC (detect circRNAs from chimeric reads), CircTest, CRAFT, ClusterProfiler, and deepTarget]. This chapter, along with a companion GitHub repository, provides a tutorial on CircRNAFlow, including installation, running the software, and interpretation of the outputs. This chapter also provides some troubleshooting tips.