Reconstructing the sequence of circular RNAs (circRNAs) from short RNA sequencing reads has proved challenging given the similarity of circRNAs and their corresponding linear messenger RNAs. Previous sequencing methods were unable to achieve high-throughput detection of full-length circRNAs. Here we describe a protocol for enrichment and full-length sequencing of circRNA isoforms using nanopore technology. Circular reverse transcription and size selection achieves a 20-fold higher enrichment of circRNAs from total RNA compared to previous methods. We developed an algorithm, called circRNA identifier using long-read sequencing data (CIRI-long), to reconstruct the sequence of circRNAs. The workflow was validated with simulated data and by comparison to Illumina sequencing as well as quantitative real-time RT–PCR. We used CIRI-long to analyze adult mouse brain samples and systematically profile circRNAs, including mitochondria-derived and transcriptional read-through circRNAs. We identified a new type of intronic self-ligated circRNA that exhibits special splicing and expression patterns. Our method takes advantage of nanopore long reads and enables unbiased reconstruction of full-length circRNA sequences.
The sequence data generated in this study have been deposited to the National Genomics Data Center54 (China National Center for Bioinformation: https://bigd.big.ac.cn/gsa) with accession number CRA003317. Details of these datasets are included in Supplementary Table 1 and the Methods section.
CIRI-long is implemented in Python and can be freely accessed at https://github.com/Kevinzjy/CIRI-long. The software is packaged with sample datasets and has been extensively tested on Linux.
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This work was supported by grants from the National Natural Science Foundation of China (32025009, 91940306, 31722031, 32071463, 91951209 and 91640117) and the National Key R&D Program (2018YFC0910400).
The authors declare no competing interests.
Peer review information Nature Biotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.
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Zhang, J., Hou, L., Zuo, Z. et al. Comprehensive profiling of circular RNAs with nanopore sequencing and CIRI-long.
Nat Biotechnol (2021). https://doi.org/10.1038/s41587-021-00842-6