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

Steffen Fuchs; Loélia Babin; Elissa Andraos; Chloé Bessiere; Semjon Willier; Johannes H. Schulte; Christine Gaspin; Fabienne Meggetto

doi:10.1371/journal.pone.0273253

Peer Review History

Original SubmissionMarch 8, 2022
23 May 2022 Decision Letter - Eduardo Andrés-León, Editor PONE-D-22-06858Generation of full-length circular RNA libraries for Oxford Nanopore long-read sequencingPLOS ONE Dear Dr. Fuchs, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Among all comments, please take into account those regarding the methodology, challenges and interpretations.In that sense, the whole protocol must be explained in detail. Please submit your revised manuscript by Jun 17 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. 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Reviewer #1: No Reviewer #2: Yes ****** 5. Is the article presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please highlight any specific errors that need correcting in the box below. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In this manuscript, the authors have developed a circRNA sequencing protocol that allows full length analysis using Oxford nanopore. Overall, the attempt to identify the full length of circRNAs is appreciated. However, the authors need to include the full detailed protocol within this manuscript with full analysis, troubleshooting, challenges, expected results and interpretations. The authors should also provide other data than those published at protocols.io with examples of fully delineated sequences of various circRNAs and confirmed by sanger sequencing. Reviewer #2: Fuchs et al. present a comprehensive protocol based on the published Full-length sequencing protocol by Zhang et al. The adapted approach differs in several aspects, such as the optimization of different circRNA lengths and addition of cleanup procedures as well as QC steps. 1. The authors mention “Modification of the ribodepletion method” as one of the parts changed in the adapted protocol. A short description on what the change is would be helpful in the abstract part of the protocol. 2. The protocol suggests use of qRT-PCR for validation of circRNAs and linear RNAs. It might be helpful to include a link to tools that can easily generate circRNA-specific primer pairs, as standard tools usually do not easily work with circular RNAs. 3. It would be helpful to directly state in the abstract on protocols.io the required amount of starting material, as this is crucial in cases where only little material is available. 4. In general, the generated data data is freely available, however, the GEO entry has an embargo date of March 2023, thus the data is not immediately available. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. 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Revision 1
25 Jun 2022 Author Response Dear Dr. Andrés-León, Thank you very much for the consideration of our manuscript and the assessment. Further, we would like to thank you for the deadline extension, which allowed us to perform a thorough review. We appreciate the comments of the two reviewers that were very helpful for us. We carefully took their concerns and recommendations into account and now provide a much more detailed protocol (version 2), including further information about the bioinformatics analysis, challenges and limitations together with expected results of this protocol and their interpretation. Further, we created new in vitro data to show the robustness of our approach by validating several circRNAs detected by Nanopore via Sanger sequencing. Changes in the manuscript are marked in blue and are italicized. We updated our protocol on protocols.io and attached a .pdf copy as supplementary file to the manuscript. The changes in the protocol we made are described in detail below, since the protocols.io website does not allow tracking of the changes. We hope that the following explanations as well as our point-by-point response to the reviewers, where we have addressed all of their concerns and comments, will make the manuscript acceptable for publication in PLOS ONE. If you require any further information, please do not hesitate to contact us directly. Thank you for your time and consideration. We look forward to hearing from you at your earliest convenience. Yours sincerely, Steffen Fuchs ---------------------- Reviewer #1: In this manuscript, the authors have developed a circRNA sequencing protocol that allows full length analysis using Oxford nanopore. Overall, the attempt to identify the full length of circRNAs is appreciated. However, the authors need to include the full detailed protocol within this manuscript with full analysis, troubleshooting, challenges, expected results and interpretations. The authors should also provide other data than those published at protocols.io with examples of fully delineated sequences of various circRNAs and confirmed by sanger sequencing. Response: We thank the reviewer for the appreciation of our provided approach to sequence circRNAs in full length and for the provided feedback and comments. We agree with the reviewer that more methodical details of the protocol, including its limitations and challenges together with troubleshooting and further more details on the expected results and their interpretation will be very helpful for the researchers planning to use it. We therefore expanded our protocol and added those details to “Steps” and added these new sections to the protocol: 5) Suggestions for Nanopore sequencing, 6) Recommendations for bioinformatics analysis of the data, 7) Expected results and interpretation, 8) Limitations and challenges, 9) Troubleshooting. Although, the creation of the sequencing libraries and the sequencing itself follows the official protocol provided by Oxford Nanopore (kits EXP-NBD104 and SQK-LSK109), we provide now explanations and suggestions for modifications that improved sequencing for us. For instance, we included an optional part on washing the flow cell during a sequencing run and reloading the library to obtain more sequencing reads. While the focus of this protocol lies on the generation of the enriched circRNA-fraction for library generation, we agree with the reviewer that more information concerning the data analysis should be provided. Therefore, we provide now detailed recommendations concerning the bioinformatics analysis including the used commands, the limitations of the analysis and how to overcome them (in the protocol at section 6) “Recommendations for bioinformatics analysis of the data” and in sections 7 to 9). This protocol is part of the materials and methods section of the manuscript and further attached as supplementary data file, now in the more detailed version 2. In the manuscript we added a summary of all this information in the abstract lines 71-73 (page 3), the introduction at lines 117-118 (page 5), and in the expected results section at lines 189-194 (page 8) and lines 236-254 (pages 9, 10). We appreciate the recommendation of the reviewer to validate circRNAs by Sanger sequencing that we detected by Oxford Nanopore. This approach will help to show the robustness of our workflow. We now selected randomly 5 circRNAs that were detected by Nanopore. The circRNAs were amplified by PCR and Sanger sequencing was performed. With this approach we could confirm all of the selected circRNAs as indicated by the detected back-splice junction in one of the anaplastic large-cell lymphoma cell lines that we sequenced by Nanopore. This information is now added as Figure 5 and supplementary figure S1 in the manuscript. Text was added to the manuscript in lines 124-140 (pages 5, 6), 217-234 (pages 8, 9). Reviewer #2: Fuchs et al. present a comprehensive protocol based on the published Full-length sequencing protocol by Zhang et al. The adapted approach differs in several aspects, such as the optimization of different circRNA lengths and addition of cleanup procedures as well as QC steps. 1. The authors mention “Modification of the ribodepletion method” as one of the parts changed in the adapted protocol. A short description on what the change is would be helpful in the abstract part of the protocol. Response: We thank the reviewer for this comment. Indeed, the ribodepletion method is crucial for the enrichment of circRNAs for the generation of libraries, since their abundance is much lower than that of ribosomal RNA. In the basic protocol from Zhang et al. the authors propose the use of a commercial ribodepletion kit (RiboErase kit, #07962266001, Kapa Biosystems). This kit, like other newer kits e.g. from New England Biolabs (NEBNext rRNA Depletion Kit v2, #E7405), is based on the protocol of Adiconis et al.[1], which uses a pool of DNA oligonucleotides directed against human rRNAs followed by a digest of DNA:RNA hybrids by RNaseH. However, the exact composition of the commercial kits remains proprietary and the used sequences are not public. In our protocol, we use the method published by Baldwin et al. [2] that is an updated and more efficient version of the Adiconis method. We use this method routinely as well successfully for the creation of Illumina RNA-sequencing libraries. Key changes are an increased ratio of DNA oligonucleotides to RNA (5:1, whereas in the Adiconis protocol a 1:1 ratio is used) and a higher incubation temperature of RNaseH (65°C in comparison to 45°C), which increases the activity and reduces the incubation time. We compared the commercial kit with the Adiconis and the Baldwin method. Adiconis and Baldwin’s methods outperformed the kit, while Baldwin’s method led to the most efficient ribodepletion. We added this information to the manuscript in line 110-112 (page 5) and in the protocol in the section “Steps”. 2. The protocol suggests use of qRT-PCR for validation of circRNAs and linear RNAs. It might be helpful to include a link to tools that can easily generate circRNA-specific primer pairs, as standard tools usually do not easily work with circular RNAs. Response: We thank the reviewer for this helpful remark. Indeed, it is important to carefully design divergent primers to specifically amplify the backsplice-junction of a circRNA without amplifying the cognate linear RNA transcribed from the same gene. We added information in the protocol to section “4) Quality control, Step 15 Validation of circRNA enrichment” on how to design primers to specifically detect circRNAs and linked two tools for primer design: CircInteractome [3] and CircPrimer 2.0 [4]. 3. It would be helpful to directly state in the abstract on protocols.io the required amount of starting material, as this is crucial in cases where only little material is available. Response: We appreciate this useful remark of the reviewer and we agree that it is crucial to mention the amount of needed starting material to prepare the sequencing libraries, especially when it comes to patient samples, which might be limited. We tested different RNA quantities to enrich for circRNAs and found that 7 µg, as we wrote in the protocol, works best and further provides enough material to sequence the samples several times to create enough data. However, we also tried 3-5 µg as input, and we could observe no major change in the amount of obtained reads, especially when multiplexing several samples, which should still lead to sufficient pore occupancy while sequencing. We added this information to the abstract and the materials section of the protocol and further in the “Expected results” part of the manuscript, lines 238-242 (page 9). 4. In general, the generated data are freely available, however, the GEO entry has an embargo date of March 2023, thus the data is not immediately available. Response: We thank the reviewer for this comment. The data was uploaded to the public repository NCBI GEO and is freely available, thus following the recommendations of PLOS ONE. However, we put an embargo on the dataset for the moment, to protect our data while the manuscript is still under revision. The data will be open as soon as the manuscript is accepted, which is in line with the NCBI GEO recommendations. We are happy to provide a temporary reviewer access to the data for the purpose of the review, which is as follows: NCBI GEO: https://www.ncbi.nlm.nih.gov/geo/, dataset ID: GSE197872, temporary reviewer password: erkpwyakjfezzed. New references added: 1. Adiconis X, Borges-Rivera D, Satija R, DeLuca DS, Busby MA, Berlin AM, et al. Comparative analysis of RNA sequencing methods for degraded or low-input samples. Nat Methods. 2013;10(7):623-9. doi: 10.1038/nmeth.2483. PubMed PMID: 23685885; PubMed Central PMCID: PMCPMC3821180. 2. Baldwin A, Morris AR, Mukherjee N. An Easy, Cost-Effective, and Scalable Method to Deplete Human Ribosomal RNA for RNA-seq. Curr Protoc. 2021;1(6):e176. Epub 2021/06/25. doi: 10.1002/cpz1.176. PubMed PMID: 34165268. 3. Dudekula DB, Panda AC, Grammatikakis I, De S, Abdelmohsen K, Gorospe M. CircInteractome: A web tool for exploring circular RNAs and their interacting proteins and microRNAs. RNA biology. 2016;13(1):34-42. doi: 10.1080/15476286.2015.1128065. PubMed PMID: 26669964; PubMed Central PMCID: PMCPMC4829301. 4. Zhong S, Wang J, Zhang Q, Xu H, Feng J. CircPrimer: a software for annotating circRNAs and determining the specificity of circRNA primers. BMC bioinformatics. 2018;19(1):292. doi: 10.1186/s12859-018-2304-1. PubMed PMID: 30075703. Attachments Attachment Submitted filename: Response to reviewers.pdf https://doi.org/10.1371/journal.pone.0273253.r002
5 Aug 2022 Decision Letter - Eduardo Andrés-León, Editor Generation of full-length circular RNA libraries for Oxford Nanopore long-read sequencing PONE-D-22-06858R1 Dear Dr. Fuchs, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Eduardo Andrés-León Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the protocol been described in sufficient detail? Descriptions of methods and reagents contained in the step-by-step protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample sizes and replication needed to ensure that the data are robust and reproducible. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Does the protocol describe a validated method? The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data. Reviewer #1: Yes Reviewer #2: Yes ****** 4. If the manuscript contains new data, have the authors made this data fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the article presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please highlight any specific errors that need correcting in the box below. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors have adressed my comments. They have expanded on the protocol, added more information regarding the method and also bioinformatics. They also have performed RT-qPCR validation. Please accept with or without changes. Reviewer #2: The authors addressed all of my points, I have no further questions. Congratulations on a very helpful protocol. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes: Kotb Abdelmohsen Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0273253.r003
Formally Accepted
26 Aug 2022 Acceptance Letter - Eduardo Andrés-León, Editor PONE-D-22-06858R1 Generation of full-length circular RNA libraries for Oxford Nanopore long-read sequencing Dear Dr. Fuchs: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Eduardo Andrés-León Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0273253.r004

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