circFL-seq, A Full-length circRNA Sequencing Method

• Published in: Bio-protocol Vol. 12; no. 8; p. e4384
• Main Authors: Liu, Zelin, Yang, Ence
• Format: Journal Article
• Language: English
• Published: United States Bio-Protocol 20.04.2022; Bio-protocol LLC
• Subjects: Biology; Methods; circFL-seq; Alternative splicing; Full-length circRNA sequencing; circRNA; Nanopore sequencing
• ISSN: 2331-8325; 2331-8325
• DOI: 10.21769/BioProtoc.4384

Due to overlapping sequences with linear cognates, identifying internal sequences of circular RNA (circRNA) remains a challenge. Recently, we have developed a full-length circRNA sequencing method (circFL-seq) and computational pipeline, to profile ordinary and fusion circRNA at the isoform level. Compared to short-read RNA-seq, rolling circular reverse transcription and nanopore long-read sequencing of circFL-seq make circRNA reads more than tenfold enriched, and show advantages for identification of both short (<100 nt) and long (>2,000 nt) circRNA transcripts. circFL-seq allows identification of differential alternative splicing suggested wide application prospects for functional studies of internal sequences in circRNAs. In addition, the experimental protocol and computational pipeline of circFL-seq shows better sensitivity and precision for identification of back-splicing junctions than current long-read sequencing methods. Together, the accurate identification and quantification of full-length circRNAs makes circFL-seq a potential tool for large-scale screening of functional circRNAs.