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

Area of Science:

• Bioinformatics and Computational Biology
• Molecular Biology and Genetics

Background:

• Circular RNAs (circRNAs) are non-coding RNAs with a unique back-splice junction (BSJ).
• Current methods for detecting circRNAs via RT-qPCR rely on specific primers, but existing design tools lack scalability, ease of use, and comprehensive feature considerations.
• Existing tools are often limited to specific organisms and do not include wet-lab validation.

Purpose of the Study:

• To develop CIRCprimerXL, a user-friendly and scalable pipeline for designing circRNA-specific RT-qPCR primers.
• To address limitations of existing tools by incorporating considerations for assay specificity, secondary structures, and SNPs.
• To enable large-scale, species-agnostic circRNA primer design.

Main Methods:

• CIRCprimerXL utilizes the Primer3 algorithm for designing BSJ-spanning primers based on circRNA BSJ positions.
• The pipeline incorporates pre-design filtering for secondary structures and common SNPs in primer annealing regions.
• Primers are further filtered for predicted specificity and amplicon secondary structures; available as a web tool and a Docker/Nextflow pipeline for broad accessibility and scalability.

Main Results:

• CIRCprimerXL successfully designed qPCR assays for over 15,000 human circRNAs, with 20 assays empirically validated.
• The Nextflow pipeline supports primer design for any species using a reference genome.
• The web tool supports primer design for human, mouse, rat, zebrafish, *Xenopus tropicalis*, and *C. elegans*.

Conclusions:

• CIRCprimerXL provides a robust, scalable, and user-friendly solution for designing high-quality circRNA-specific RT-qPCR primers.
• The tool overcomes limitations of previous pipelines by considering crucial design parameters and supporting multiple species.
• CIRCprimerXL facilitates efficient circRNA research through automated, validated primer design.