circFL-seq reveals full-length circular RNAs with rolling circular reverse transcription and nanopore sequencing

Abstract

Circular RNAs (circRNAs) act through multiple mechanisms via their sequence features to fine-tune gene expression networks. Due to overlapping sequences with linear cognates, identifying internal sequences of circRNAs remains a challenge, which hinders a comprehensive understanding of circRNA functions and mechanisms. Here, based on rolling circular reverse transcription (RCRT) and nanopore sequencing, we developed circFL-seq, a full-length circRNA sequencing method, to profile circRNA at the isoform level. With a customized computational pipeline to directly identify full-length sequences from rolling circular reads, we reconstructed 77,606 high-quality circRNAs from seven human cell lines and two human tissues. circFL-seq benefits from rolling circles and long-read sequencing, and the results showed more than tenfold enrichment of circRNA reads and advantages for both detection and quantification at the isoform level compared to those for short-read RNA sequencing. The concordance of the RT-qPCR and circFL-seq results for the identification of differential alternative splicing suggested wide application prospects for functional studies of internal variants in circRNAs. Moreover, the detection of fusion circRNAs at the omics scale may further expand the application of circFL-seq. Together, the accurate identification and quantification of full-length circRNAs make circFL-seq a potential tool for large-scale screening of functional circRNAs.

Emerging Roles of Circular RNAs in Vascular Smooth Muscle Cell Dysfunction

“…For shotgun sequencing-based circRNA profiling, the additional procedures usually include linear RNA removal through exonuclease digestion coupled with identification of back-spliced reads using specific bioinformatic programmes, such as CIRI2 ( Gao et al, 2018 ), DCC ( Cheng et al, 2016 ), Sailfish-cir ( Li et al, 2017 ) and CIRIquant ( Zhang et al, 2020 ), each of which has distinct sensitivity, reliability, and computational requirement. Aside from shotgun sequencing, a newer approach based on rolling circular reverse transcription and nanopore sequencing is available for the annotation of the full repertoires of circRNAs ( Liu et al, 2021a ). Microarrays with probes that target back-splice sites have also been widely used for circRNA profiling ( Li et al, 2019b ).…”

Promising Roles of Circular RNAs as Biomarkers and Targets for Potential Diagnosis and Therapy of Tuberculosis

“…Therefore, improved experimental strategies and computational tools for the accurate identification of the full length of circRNAs are needed. Recently, many full-length circRNAs sequencing methods such as isoCirc [ 44 ], CIRI-long [ 45 ], circNick-LRS [ 46 ], circFL-seq [ 47 ] have been developed to profile circRNAs at the isoform level. IsoCirc utilizes the rolling loop amplification and Oxford Nanopore sequencing to detect the full length of circRNAs and their internal splicing structures, overcoming the defect of short-read RNA sequencing [ 44 ].…”

“…CircFL-seq can detect the full length of circRNAs by rolling circular reverse transcription (RCRT) and nanopore long-read sequencing, which makes circRNAs reads more than ten-fold enriched compared to short-read RNA-seq. Moreover, as opposed to isoCirc and CIRI-long, circFL-seq adds poly(A) tail after RCRT and then the circRNAs sequences are amplified by adding primer connectors and captured variable isomers accurately, which provides it with the ability to identify more variable splicing events [ 47 ]. However, limitations on these emerging techniques should be acknowledged.…”

“…Simultaneously, some circRNAs can also be digested by RNase R enzyme, which causes the circRNAs’ library to deviate from the fact. Another problem is that the sequencing depth of IsoCirc and CIRI-long is not enough, and the detection rate of circRNA will continue to rise with deeper sequencing [ 44 , 45 , 46 , 47 ].…”

Characterization of circular RNAs with advanced sequencing technologies in human complex diseases

“…In 2015, the TGS method was already applied to characterize a handful of full-length circRNAs in mouse brains (You et al, 2015). While more recently, in 2021, four research groups utilized the state-of-art high throughput Nanopore sequencing technology to comprehensively characterize full-length circRNAs (Liu et al, 2021;Rahimi et al, 2021;Xin et al, 2021;. With longer circRNAs discovered, new molecular processes were explored.…”