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

Abstract

Circular RNAs (circRNAs) act through multiple mechanisms with their sequence features to fine-tune gene expression networks. Due to overlapping sequences with linear cognates, identifying internal sequences of circRNAs remains a great challenge, which hinders 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 circfull 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. Benefiting from rolling circles and long-read sequencing, circFL-seq showed more than tenfold enrichment of circRNA reads and advantages for both detection and quantification at the isoform level compared to short-read RNA sequencing. The concordance of 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 cancer-related 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.

Assessing the impacts of various factors related to identification, conservation, biogenesis, and function on circular RNA reliability

“…2C). Since the performance of CIRI-full, a short read-based approach, for identifying full-length sequences of circRNAs is hampered by the length of short reads (Zheng et al 2019), we also extracted the full-length circRNA candidates identified by circFL-seq based on nanopore long reads (Liu et al 2021). Such a long read-based approach provided the direct support of full-length circular sequences from single-molecule long reads.…”

“…For the most important factor in affecting circRNA reliability, since template switching events occur randomly during reverse transcription in vitro , a circRNA candidate observed in multiple samples or replicates is less likely to be generated from such an in vitro artifact. For the second most important factor in affecting circRNA reliability, full-length circular sequences can be reconstructed by Illumina short RNA-seq reads with the reverse overlap (RO) feature (Zheng et al 2019) or identified by single-molecule long reads (Liu et al 2021). With the support of RO-merged reads, the BSJ events are less probable to originate from template switching because both ends of the paired-end reads are sequencing from the same fragment and reversely overlapped with each other by alignment.…”

“…The factors of CIRI-full-identified full-length circular sequence, number of circRNA detection tools, conservation patterns at the individual (or sample), tissue, and specie levels (including tissue specificity index) were extracted from circAtlas 2.0 (Wu et al 2020). The BSJ events with the direct support of full-length circular sequences from nanopore long RNA-seq reads were downloaded from Liu et al’s study (Liu et al 2021). The evolutionary rates were determined by phyloP (Pertea et al 2011) or phastCons (Siepel et al 2005) scores, which were downloaded from the UCSC Genome Browser at https://genome.ucsc..edu.…”

Circular RNAs: Biomarkers of cancer

“…Compared with next‐generation sequencing, the optimized nanopore sequencing system and bioinformatics methods can greatly improve the detection efficiency and accurately quantify the expression of circRNAs. Moreover, nanopore sequencing can achieve the full sequence assembly of circRNAs with a length of 100 bp–5 kb [50–53]. Real‐time quantitative polymerase chain reaction and Northern blot are the primary methods used to analyze circRNA expression.…”

HIV-1 Vpr induces ciTRAN to prevent transcriptional silencing of the provirus

“…Moreover, PCR bias can lead to more efficient amplification of some circRNAs, additional events such as trans-splicing and exon repetitions further hamper identification of circRNA in the native form(13). Accordingly, the long-read sequencing technology has been applied to circRNAs only from reverse transcribed products (14–16).…”