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

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.

Discovery and Comprehensive Characterization of Novel Circular RNAs of the Apoptosis-Related BOK Gene in Human Ovarian and Prostate Cancer Cells, Using Nanopore Sequencing

“…Additionally, the use of exon skipping and extended BOK exons in the novel cir-cRNA structure was also prevalent in our dataset. These events are in agreement with similar studies that report alternative splicing events in circRNAs [43,44]. Interestingly, the validation of the existence of the non-coding BOK exon between the first and second coding exons-which so far has been an outcome of computational analysis of expressed sequence tags (ESTs)-is evidenced by our results, considering that it participates in the structure of three novel circRNAs either as a full-length exon or as a 5 -truncated one.…”

Defining the landscape of circular RNAs in neuroblastoma unveils a global suppressive function of MYCN

“…The median length of all detected circRNAs predicted by short-read sequencing was 844 nt (728 nt when only considering exonic circRNAs). We aimed to evaluate this estimation by using our recently developed protocol for the Oxford Nanopore long-read sequencing platform to sequence circRNAs in full length 21 . We sequenced six different neuroblastoma cell lines and detected a shorter median length of 474 nt (Supplementary Fig.…”

“…Oxford Nanopore long-read sequencing was used to analyze circRNAs in full-length from 6 different neuroblastoma cell lines: IMR-5, LS, SH-EP, SH-SY5Y, Lan-5, SK-N-FI. Sequencing was performed as described before 21 with a MinION MK1C sequencer using the MinKNOW software (v22.05.8). A detailed and fully referenced protocol was deposited on protocols.io, 10.17504/protocols.io.rm7vzy8r4lx1/v2.…”

Current advances in circular RNA detection and investigation methods: Are we running in circles?

“…The protocol presumably can be completed in less than a day and 1 ug of total RNA is sufficient for library construction (Hou et al, 2023). Modifications of the CIRI-long protocol have been developed, such as one based on the replacement of the commercial kit used for ribodepletion with a method utilizing a pool of DNA oligonucleotides that bind and hybridize the rRNA, making it susceptible to degradation by RNase H (Baldwin et al, 2021), deeming it more efficient, scalable, and cost-effective (Fuchs et al, 2022).…”

“…In addition, nanopore sequencing can detect and quantify low abundance circRNAs, which is an important aspect as many circRNAs are present at low levels in biological samples (Lu et al, 2021). Full‐length reconstruction has been accomplished using Illumina and Nanopore RNA‐seq datasets (Fuchs et al, 2022). Nanopore sequencing library preparation have adopted rolling‐circle amplification (RCA)‐based methods, such as IsoCirc, for the construction of circular cDNA concatemers that act as enriched templates (Xin et al, 2021).…”