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Comprehensive profiling of circular RNAs with nanopore sequencing and CIRI-long

Jinyang Zhang, Lingling Hou, Zhenqiang Zuo, Peifeng Ji, Xiaorong Zhang, Yuanchao Xue,
Published: 11 March 2021
Nature Biotechnology pp 1-10; doi:10.1038/s41587-021-00842-6

Abstract: 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 rich diversity of circular RNAs is sequenced using nanopore long reads.
Keywords: RNA sequencing / sequencing reads / full length circRNAs / using nanopore / reconstruct the sequence

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