IsoCirc catalogs full-length circular RNA isoforms in human transcriptomes

Ruijiao Xin,Robert Wang,Yuan Gao,Bo Liu,Yi Xing,Yan Gao,Yadong Wang,Lan Lin,Kathryn E Kadash-Edmondson

doi:10.1038/s41467-020-20459-8

Abstract

Circular RNAs (circRNAs) have emerged as an important class of functional RNA molecules. Short-read RNA sequencing (RNA-seq) is a widely used strategy to identify circRNAs. However, an inherent limitation of short-read RNA-seq is that it does not experimentally determine the full-length sequences and exact exonic compositions of circRNAs. Here, we report isoCirc, a strategy for sequencing full-length circRNA isoforms, using rolling circle amplification followed by nanopore long-read sequencing. We describe an integrated computational pipeline to reliably characterize full-length circRNA isoforms using isoCirc data. Using isoCirc, we generate a comprehensive catalog of 107,147 full-length circRNA isoforms across 12 human tissues and one human cell line (HEK293), including 40,628 isoforms ≥500 nt in length. We identify widespread alternative splicing events within the internal part of circRNAs, including 720 retained intron events corresponding to a class of exon-intron circRNAs (EIciRNAs). Collectively, isoCirc and the companion dataset provide a useful strategy and resource for studying circRNAs in human transcriptomes.

Highlights

Circular RNAs have emerged as an important class of functional RNA molecules
From total RNAs extracted from a biological sample, circRNAs are enriched and linear RNAs are depleted through ribosomal RNA removal and RNase R treatment
The reverse transcription (RT) product is ligated into a circular cDNA, which is amplified through rolling circle amplification (RCA)

Summary

Introduction

Circular RNAs (circRNAs) have emerged as an important class of functional RNA molecules. By analyzing RNA-seq data of multiple human cell types, Salzman and colleagues provided the first evidence for the widespread occurrence of circRNA BSJs in the human transcriptome They observed that circRNAs are the predominant transcript isoform in hundreds of human genes[14]. Inferring the protein products translated from circRNAs requires full-length circRNA sequences[11] To fill this gap, several computational methods have been developed to reconstruct full-length circRNAs from short-read RNA-seq data[25,26,27,28,29]. Long-read sequencing platforms such as Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) can generate reads that are thousands to tens of thousands of bases in length, making long-read RNA-seq a powerful tool for resolving full-length transcript isoforms In this manuscript, we report isoCirc, a strategy for sequencing full-length circRNA isoforms, using rolling circle amplification (RCA)

Methods

Results

Conclusion