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

Cytidine-to-Uridine (C-to-U) RNA editing is a post-transcriptional modification essential for various biological processes. APOBEC deaminases mediate C-to-U editing which play critical role in cellular function and regulation. Advances in next-generation sequencing (NGS) technologies and analytical tools have provided powerful means to assess RNA editing activities and their physiological implications. However, inherent errors in NGS workflows-including reverse transcription, PCR amplification, and sequencing-complicate the detection of actual editing events. With error rates ranging from 10^-2 to 10^-3 per nucleotide, these technical artifacts can obscure APOBEC-mediated editing events occurring at similar frequencies. To address these challenges, in this chapter, we describe two established and optimized RNA sequencing strategies explicitly designed to detect low-frequency RNA editing events accurately while distinguishing them from NGS-associated errors. These methods are termed "circular RNA Sequencing Assay" and "Safe-Sequencing System (SSS)" and enable the reliable identification of RNA editing events (and also somatic mutations) at or below typical error thresholds.