What is Circular RNA Sequencing (circRNA-Seq)?

Circular RNA Sequencing (circRNA-Seq) is a specialized RNA sequencing technique designed to detect and quantify circular RNAs (circRNAs). Unlike linear RNA, circRNAs are covalently closed-loop structures that do not have 5′ caps or 3′ tails. These molecules are formed by back-splicing events and are highly stable, playing significant roles in gene regulation, disease development, and possibly protein translation.

circRNA-Seq enables the identification, quantification, and functional annotation of these unique RNA molecules across different biological conditions.

Overview

• Focus: Detection and analysis of circular RNAs

• Structure of circRNAs: Closed-loop, non-polyadenylated, resistant to exonucleases

• Origin: Derived from pre-mRNA via back-splicing

• Relevance: Regulate gene expression, act as miRNA sponges, potential biomarkers

• Technology Used: Next-Generation Sequencing (NGS)

How circRNA Sequencing Works

1. Total RNA Extraction
   Total RNA is isolated from the sample.

2. Ribosomal RNA (rRNA) Removal
   Ribosomal RNA is depleted since it dominates total RNA.

3. Linear RNA Degradation (optional)
   RNase R enzyme is often used to digest linear RNAs while preserving circular RNAs.

4. Library Preparation
   circRNAs are reverse-transcribed into cDNA and sequencing adapters are added.

5. Sequencing
   Libraries are sequenced using paired-end sequencing to capture back-splice junctions.

6. Bioinformatics Analysis
   Specialized tools detect non-canonical splice junctions that indicate circular RNA presence.

Applications of circRNA-Seq

• Cancer Research
  Detects circRNAs that act as oncogenes or tumor suppressors.

• Neurodegenerative Diseases
  Studies show circRNAs are enriched in brain tissues and linked to diseases like Alzheimer’s.

• Biomarker Discovery
  circRNAs are stable in blood and tissue, making them strong non-invasive biomarkers.

• miRNA Sponge Identification
  Many circRNAs bind and sequester miRNAs, regulating gene expression.

• Transcriptome Profiling
  Complements traditional RNA-seq by revealing additional regulatory RNAs.

Types of circRNAs Identified

Advantages of circRNA Sequencing

• High Stability Detection: circRNAs are naturally resistant to degradation.

• Specificity: Detects back-splicing events unique to circRNAs.

• High Resolution: Identifies isoforms and tissue-specific circRNA expression.

• Biomarker Potential: Ideal for non-invasive diagnostic research.

• Quantitative: Enables expression-level comparisons between conditions.

Limitations and Challenges

• Low Abundance: circRNAs are often less abundant than linear RNAs.

• Data Complexity: Requires specialized algorithms to identify back-splice junctions.

• Experimental Design: Enrichment steps like RNase R digestion must be carefully optimized.

• Annotation Limitations: Reference databases for circRNAs are still evolving.

Bioinformatics Tools for circRNA Analysis

• CIRCexplorer2 – Identifies circRNAs and annotates back-splice junctions.

• find_circ – Lightweight tool for circRNA detection from RNA-seq reads.

• DCC (Detection of circRNAs) – Handles paired-end sequencing and complex filtering.

• circBase – A widely-used circRNA database for annotation and reference.

• CIRIquant – Combines quantification with statistical modeling for expression analysis.

Who Uses circRNA-Seq?

• Molecular Biologists – To explore new layers of gene regulation.

• Cancer Genomics Researchers – Investigating novel tumor biomarkers.

• Bioinformaticians – Developing models and visualizations for RNA networks.

• Clinical Scientists – Seeking stable RNA biomarkers in fluids like blood, saliva, etc.

• Neurologists – Studying circRNA expression in aging and neurodegeneration.

Comparison: circRNA-Seq vs Other RNA-Seq Methods