Low-error RNA sequencing techniques for detecting RNA editing by APOBECs: Circular RNAseq assay and safe-sequencing...

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Cytidine-to-Uridine (C-to-U) RNA editing is a vital post-transcriptional modification mediated by APOBEC deaminases.
• This editing plays a critical role in cellular function and regulation.
• Next-generation sequencing (NGS) advances enable RNA editing assessment but face challenges from inherent workflow errors.

Purpose of the Study:

• To describe optimized RNA sequencing strategies for accurate detection of low-frequency RNA editing events.
• To differentiate genuine RNA editing from technical artifacts in NGS data.
• To enable reliable identification of RNA editing and somatic mutations below typical error thresholds.

Main Methods:

• Description of two established and optimized RNA sequencing strategies: circular RNA Sequencing Assay and Safe-Sequencing System (SSS).
• These methods are designed to minimize and distinguish NGS-associated errors from true editing events.
• Focus on accurate detection of low-frequency editing events.

Main Results:

• The described methods enable accurate detection of RNA editing events.
• These strategies effectively distinguish genuine editing from NGS errors (e.g., reverse transcription, PCR, sequencing artifacts).
• Reliable identification of RNA editing and somatic mutations at or below typical error thresholds is achieved.

Conclusions:

• Circular RNA Sequencing Assay and Safe-Sequencing System (SSS) are effective for detecting low-frequency RNA editing.
• These methods overcome limitations posed by NGS technical errors.
• Accurate RNA editing detection is crucial for understanding its role in cellular function and disease.