Generation of full-length circular RNA libraries for Oxford Nanopore long-read sequencing

PLoS One. 2022 Sep 7;17(9):e0273253. doi: 10.1371/journal.pone.0273253. eCollection 2022.

Abstract

Circular RNA (circRNA) is a noncoding RNA class with important implications for gene expression regulation, mostly by interaction with other RNA species or RNA-binding proteins. While the commonly applied short-read Illumina RNA-sequencing techniques can be used to detect circRNAs, their full sequence is not revealed. However, the complete sequence information is needed to analyze potential interactions and thus the mechanism of action of circRNAs. Here, we present an improved protocol to enrich and sequence full-length circRNAs by using the Oxford Nanopore long-read sequencing platform. The protocol involves an enrichment of lowly abundant circRNAs by exonuclease treatment and negative selection of linear RNAs. Then, a cDNA library is created and amplified by PCR. This protocol provides enough material for several sequencing runs. The library is used as input for ligation-based sequencing together with native barcoding. Stringent quality control of the libraries is ensured by a combination of Qubit, Fragment Analyzer and qRT-PCR. Multiplexing of up to 4 libraries yields in total more than 1-2 Million reads per library, of which 1-2% are circRNA-specific reads with >99% of them full-length. The protocol works well with human cancer cell lines. We further provide suggestions for the bioinformatic analysis of the created data, as well as the limitations of our approach together with recommendations for troubleshooting and interpretation. Taken together, this protocol enables reliable full-length analysis of circRNAs, a noncoding RNA type involved in a growing number of physiologic and pathologic conditions. Metadata Associated content. https://dx.doi.org/10.17504/protocols.io.rm7vzy8r4lx1/v2.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • High-Throughput Nucleotide Sequencing / methods
  • Humans
  • Nanopores*
  • RNA / genetics
  • RNA, Circular*
  • Sequence Analysis, RNA / methods

Substances

  • RNA, Circular
  • RNA

Grants and funding

S.F. is a participant in the BIH-Charité Clinician Scientist Program funded by the Charité – Universitätsmedizin Berlin and the Berlin Institute of Health. S.F. was supported during work by a grant from the Berliner Krebsgesellschaft e.V. (grant no. FUFF201721KK), the Stiftung Tumorforschung Kopf-Hals (Wiesbaden, Germany) and the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, grant no. 439441203). F.M was supported by grants from Inserm, l’association Eva pour la vie, the Federation Grandir Sans Cancer and La ligue contre le cancer (Equipes Labelisée 2017-2021). E.A. is supported by a grant from Labex TOUCAN/Laboratoire d’excellence Toulouse Cancer. L.B. and C.B. are supported by fellowships from the Fondation de France. The funders had and will not have a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.