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Full-length circular RNA profiling by nanopore sequencing with CIRI-long

Nature Protocols volume 18, pages 1795–1813 (2023)Cite this article

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Abstract

Circular RNAs (circRNAs) have important roles in regulating developmental processes and disease progression. As most circRNA sequences are highly similar to their cognate linear transcripts, the current short-read sequencing-based methods rely on the back-spliced junction signal for distinguishing circular and linear reads, which does not allow circRNAs’ full-length structure to be effectively reconstructed. Here we describe a long-read sequencing-based protocol, CIRI-long, for the detection of full-length circular RNAs. The CIRI-long protocol combines rolling circular reverse transcription and nanopore sequencing to capture full-length circRNA sequences. After poly(A) tailing, RNase R treatment, and size selection of polymerase chain reaction products, CIRI-long achieves an increased percentage (6%) of circular reads in the constructed library, which is 20-fold higher compared with previous Illumina-based strategies. This method can be applied in cell lines or tissue samples, enabling accurate detection of full-length circRNAs in the range of 100–3,000 bp. The entire protocol can be completed in 1 d, and can be scaled up for large-scale analysis using the nanopore barcoding kit and PromethION sequencing device. CIRI-long can serve as an effective and user-friendly protocol for characterizing full-length circRNAs, generating direct and convincing evidence for the existence of detected circRNAs. The analytical pipeline offers convenient functions for identification of full-length circRNA isoforms and integration of multiple datasets. The assembled full-length transcripts and their splicing patterns provide indispensable information to explore the biological function of circRNAs.

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Fig. 1: Overview of the CIRI-long protocol.
Fig. 2: Applications of the CIRI-long protocol.
Fig. 3: RNA hydrolysis and TS during circular RT.
Fig. 4: Overview of the CIRI-long data analysis workflow.
Fig. 5: Anticipated library size after beads selection.

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Data availability

The CIRI-long dataset used in this manuscript can be downloaded from the National Genomics Data Center46 (China National Center for Bioinformation: https://bigd.big.ac.cn/gsa) with accession number CRR194209.

Code availability

The CIRI-long software can be downloaded from GitHub at https://github.com/bioinfo-biols/CIRI-long47.

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Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (32130020, 32025009, 91940306, 32200530) and the National Key R&D Project (2021YFA1300500, 2021YFA1302000).

Author information

Author notes

  1. These authors contributed equally: Lingling Hou, Jinyang Zhang.

Authors and Affiliations

  1. Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China

    Lingling Hou, Jinyang Zhang & Fangqing Zhao

  2. University of Chinese Academy of Sciences, Beijing, China

    Fangqing Zhao

  3. Key Laboratory of Systems Biology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Hangzhou, China

    Fangqing Zhao

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Contributions

L.H. and J.Z. developed the experimental protocol for CIRI-long. L.H., J.Z. and F.Z. wrote the manuscript. F.Z. conceived the study.

Corresponding authors

Correspondence to Jinyang Zhang or Fangqing Zhao.

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Nature Protocols thanks Michael Clark and Leng Han for their contribution to the peer review of this work.

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Key reference using this protocol

Zhang, J. et al. Nat. Biotechnol. 39, 836–845 (2021): https://doi.org/10.1038/s41587-021-00842-6

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Hou, L., Zhang, J. & Zhao, F. Full-length circular RNA profiling by nanopore sequencing with CIRI-long. Nat Protoc 18, 1795–1813 (2023). https://doi.org/10.1038/s41596-023-00815-w

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