Abstract
Circular RNAs (circRNAs) act through multiple mechanisms via their sequence features to fine-tune gene expression networks. Due to overlapping sequences with linear cognates, identifying internal sequences of circRNAs remains a challenge, which hinders a comprehensive understanding of circRNA functions and mechanisms. Here, based on rolling circular reverse transcription and nanopore sequencing, we developed circFL-seq, a full-length circRNA sequencing method, to profile circRNA at the isoform level. With a customized computational pipeline to directly identify full-length sequences from rolling circular reads, we reconstructed 77,606 high-quality circRNAs from seven human cell lines and two human tissues. circFL-seq benefits from rolling circles and long-read sequencing, and the results showed more than tenfold enrichment of circRNA reads and advantages for both detection and quantification at the isoform level compared to those for short-read RNA sequencing. The concordance of the RT-qPCR and circFL-seq results for the identification of differential alternative splicing suggested wide application prospects for functional studies of internal variants in circRNAs. Moreover, the detection of fusion circRNAs at the omics scale may further expand the application of circFL-seq. Taken together, the accurate identification and quantification of full-length circRNAs make circFL-seq a potential tool for large-scale screening of functional circRNAs.
Highlights
CircRNAs, a class of covalently closed RNA molecules formed via back-splicing (BS)or lariat precursors, are involved in various biological processes and pathogenesis by 42 fine-tuning the eukaryotic gene regulatory network[1,2]
We developed the circFL-seq approach that utilized reverse transcription (RCRT) with long-read sequencing for full-length circRNA profiling (Figure 1A)
First-strand cDNA was synthesized with random primers (P1-N6) by RCRT for circRNA and regular reverse transcription for linear residuals
Summary
CircRNAs, a class of covalently closed RNA molecules formed via back-splicing (BS). or lariat precursors, are involved in various biological processes and pathogenesis by 42 fine-tuning the eukaryotic gene regulatory network[1,2]. By detecting the back-splicing junctions (BSJs) of circRNAs with deep sequencing, short-read RNA sequencing discriminates circRNAs with low expression 52 (as low as 1% polyadenylated RNA9) from their linear cognates. Single-molecule long-read sequencing has shown methodological advances in identifying circRNAs at the isoform level. We developed a high-throughput circRNA sequencing method, termed circFL-seq, with rolling circular reverse transcription[16,17] (RCRT) and nanopore long-read sequencing for the identification of circRNA at the isoform level. By providing full-length circRNA sequences, circFL-seq allowed the study of sequence features, alternative splicing, and differential expression at the isoform level. CircFL-seq benefits from RCRT and long-read sequencing and has shown advantages for identifying high-quality full-length circRNAs at a low cost
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