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  1. Evaluation Summary:

    This study provides an experimental and computational method for the identification and reconstruction of full-length circRNAs using nanopore sequencing,. With a better comparison to other existing methods in the field and a clearer demonstration of the advantages of the described methodology, the work would be of great interest to researchers in the circular RNA community.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    Ence Yang and colleagues report circFL-seq, a method for sequencing full-length circular RNAs with rolling circle reverse transcription and nanopore sequencing. The authors carried out comprehensive computational analyses and experimental validations of circFL-seq data, and reported novel biological findings (e.g. fusion circular RNAs).

    Overall, this paper will be a valuable addition to a growing body of literature on long read sequencing of circular RNAs. A major strength of this work is that it combines comprehensive computational analyses with rigorous experimental validations. Specifically, the authors assessed circFL-seq results using short-read RNA-seq data as well as RT-PCR experiments. They also honed into a specific type of novel circular RNAs (i.e. fusion circular RNAs), and demonstrated that circFL-seq discovery of fusion circular RNAs can be validated by RT-PCR. Additionally, the authors compared circFL-seq data to data generated by isoCirc, a recently published method for sequencing full-length circular RNAs, that combines rolling circle amplification followed by nanopore sequencing.

    A notable weakness of the present manuscript is with the comparison to existing methods. While the authors compared circFL-seq to isoCirc, they only cited CIRI-long but didn't compare circFL-seq to CIRI-long. Given that both circFL-seq and CIRI-long are based on rolling circle reverse transcription while isoCirc is based on rolling circle amplification, a three-way comparison would have been quite informative. Additionally, given that all three methods were developed independently at almost the same time, the authors would need to provide a more objective discussion about the strengths and weaknesses of individual methods. The current manuscript appears to present circFL-seq as a more superior method for nanopore sequencing of full-length circular RNAs, but the claims and conclusions are not sufficiently supported by their data and the current literature.

  3. Reviewer #2 (Public Review):

    In the manuscript entitled "circFL-seq reveals full-length circular RNAs with rolling circular reverse transcription and nanopore sequencing", the authors develop a technology combining RCRT and nanopore long-read sequencing to identify the circRNA isoforms in cell lines and human tissues. The authors claim that the circFL-seq gives the advantages of detection and quantification of circRNA isoforms in comparison to short-read sequencing. The authors also claim that circFL-seq has the advantages of less linear RNA residual contamination and more cost-efficient than RCA long-read sequencing. Finally, the authors can detect fusion circRNAs in MCF7 cells with circFL-seq. Nevertheless, as pointed out by the authors themselves, similar approaches of detecting circRNA isoforms using long-read nanopore sequencing already exists (eg. isoCirc); hence it is the authors' burden to prove that circFL-seq has advantages over isoCirc. However, the authors do not provide enough evidence showing that RCRT indeed gives less linear RNA residual contamination. Moreover, the fact that circFL-seq identifies fewer circRNA isoforms than isoCirc also raises a flag of possible low capture efficiency of RCRT (see major concerns below). In addition, while discovering full length circRNAs using long-read sequencing is interesting, the authors do not provide data suggesting the biological relevance of these identified transcripts. Overall, the authors would would need to provide more data and findings to really strengthen the novelty and the significance of the manuscript.

  4. Reviewer #3 (Public Review):

    In the manuscript entitled "circFL-seq reveals full-length circular RNAs with rolling circular reverse transcription and nanopore sequencing", the authors developed a novel strategy to use nanopore sequencing to profile circRNA at the isoform level. Compared with previous short-read RNA sequencing methods, circFL-seq largely increased the sensitivity of circRNA detection. The authors applied circFL-seq to seven human cell lines and two human tissues and discovered novel fusion circRNAs that might play important roles in cancer. The manuscript is well written and the conclusions are well supported by their analyses.


    In two recently published studies, isoCirc (PMID: 33436621) and CIRI-long (PMID: 33436621) have also used nanopore sequencing to characterize circRNA isoforms and alternative splicing events. It would have been good to see a detailed comparisin between these studies and the current work. For example, both studies have reported a relatively higher percentage of retained introns (isoCirc: Fig. 4b, CIRI-long: Supplementary Fig. 13) compared to the number of 3.5% of intron retention events in line 139. It would be helpful if the authors could clarify the reason behind this difference.