CIRCprimerXL: Convenient and High-Throughput PCR Primer Design for Circular RNA Quantification

Circular RNA (circRNA) is a class of endogenous non-coding RNA characterized by a back-splice junction (BSJ). In general, large-scale circRNA BSJ detection is performed based on RNA sequencing data, followed by the selection and validation of circRNAs of interest using RT-qPCR with circRNA-specific PCR primers. Such a primer pair is convergent and functional on the circRNA template but divergent and non-functional on the linear host gene. Although a few circRNA primer design pipelines have been published, none of them offer large-scale, easy-to-use circRNA primer design. Other limitations are that these tools generally do not take into account assay specificity, secondary structures, and SNPs in the primer annealing regions. Furthermore, these tools are limited to circRNA primer design for humans (no other organisms possible), and no wet-lab validation is demonstrated. Here, we present CIRCprimerXL, a circRNA RT-qPCR assay design pipeline based on the primer design framework primerXL. CIRCprimerXL takes a circRNA BSJ position as input, and designs BSJ-spanning primers using Primer3. The user can choose to use the unspliced or spliced circRNA sequence as template. Prior to primer design, sequence regions with secondary structures and common SNPs are flagged. Next, the primers are filtered based on predicted specificity and the absence of secondary structures of the amplicon to select a suitable primer pair. Our tool is both available as a user-friendly web tool and as a stand-alone pipeline based on Docker and Nextflow, allowing users to run the pipeline on a wide range of computer infrastructures. The CIRCprimerXL Nextflow pipeline can be used to design circRNA primers for any species by providing the appropriate reference genome. The CIRCprimerXL web tool supports circRNA primer design for human, mouse, rat, zebrafish, Xenopus tropicalis, and C. elegans. The design process can easily be scaled up for the qPCR assay design of tens of thousands of circRNAs within a couple of hours. We show how CIRCprimerXL has been successfully used to design qPCR assays for over 15,000 human circRNAs of which 20 were empirically validated. CIRCprimerXL software, documentation, and test data can be found at: https://github.com/OncoRNALab/CIRCprimerXL. CIRCprimerXL is also implemented as a webtool at: https://circprimerxl.cmgg.be.