Full-length circular RNA profiling by nanopore sequencing with CIRI-long

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• Circular RNAs (circRNAs) play key roles in biological processes and disease.
• Existing short-read sequencing methods struggle to reconstruct full-length circRNA structures due to sequence similarity with linear transcripts.
• Distinguishing circular from linear reads relies on back-spliced junction signals, limiting comprehensive analysis.

Purpose of the Study:

• To develop a novel long-read sequencing protocol for the detection and characterization of full-length circular RNAs.
• To overcome the limitations of short-read sequencing in circRNA structure reconstruction.
• To provide a user-friendly and efficient method for exploring circRNA biological functions.

Main Methods:

• The CIRI-long protocol integrates rolling circular reverse transcription and nanopore sequencing.
• Key steps include poly(A) tailing, RNase R treatment, and size selection of PCR products.
• The method is applicable to cell lines and tissue samples for detecting circRNAs between 100-3,000 bp.

Main Results:

• CIRI-long achieved a 6% yield of circular reads, a 20-fold increase compared to previous Illumina-based strategies.
• The protocol enables the accurate detection of full-length circRNAs.
• The entire process can be completed within 1 day and scaled for large-throughput analysis.

Conclusions:

• CIRI-long offers an effective and user-friendly approach for characterizing full-length circRNAs.
• This method provides direct evidence for circRNA existence and facilitates the exploration of their biological functions.
• The analytical pipeline supports the identification of circRNA isoforms and integration of diverse datasets.