RespiCoV: Simultaneous identification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and 46 respiratory tract viruses and bacteria by amplicon-based Oxford-Nanopore MinION sequencing

Annika Brinkmann; Steven Uddin; Sophie-Luisa Ulm; Katharina Pape; Sophie Förster; Khalid Enan; Jalal Nourlil; Eva Krause; Lars Schaade; Janine Michel; Andreas Nitsche

doi:10.1371/journal.pone.0264855

Peer Review History

Original SubmissionOctober 8, 2021
7 Dec 2021 Decision Letter - Kok Keng Tee, Editor PONE-D-21-32371RespiCoV: Simultaneous identification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and 40 respiratory tract viruses by amplicon-based Oxford-Nanopore MinION sequencingPLOS ONE Dear Dr. Brinkmann, 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. Please submit your revised manuscript by Jan 15 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. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: N/A ****** 3. Have the authors made all data underlying the findings in their manuscript 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: No ****** 4. Is the manuscript 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 note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. 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 describe a method based on the combination of multiplex PCR with MinION sequencing for the simultaneous detection of SARS-CoV-2 and a panel of potential co-infecting agents. The study was well conducted and no major issues were identified. Some minor comments are provided below. Please provide additional information on how the species identification was performed upon acquisition of the reads. Use the term "Cq" instead of "Ct" according to Bustin et al., throughout the manuscript, including in the tables. In line 161 a "." seems to be missing in the Cq value. Whenever referring to Tables and Figures, start with with capital "T" and/ or "F". Tables should be improved, provide captions with additional information. With the data obtained can the authors provide details regarding the analytical sensitivity, specificity and accuracy of their method? Reviewer #2: Brinkmann and colleagues presented and evaluated an alternative method for simultaneous identification of respiratory pathogens using amplicon sequencing on a nanopore platform. The method was evaluated with clinical samples and compared with commercially available multiplex screening assays. In addition, the methodology was tested in an international quality assurance exercise. The study is interesting and valuable because respiratory pathogens other than SARS-CoV-2 are often undiagnosed during the pandemic. Sequencing using the Nanopore platform is also becoming more available worldwide. The manuscript is concise and clearly written, although some minor improvements should be made. Title: the proposed panel also is intended to detect some bacterial pathogens. It would be good to adjust the title accordingly to indicate the broader scope of this method. Data access statement: the authors state that all data are available through the SRA databases. The accession number should be included in the manuscript. Bioinformatics analysis: the author used the standard software provided by the sequencing company, which could be advantageous in a clinical setting, but the authors may wish to compare their results with more advanced workflows available for nanopore reads. Please provide further details on the primer trimming method. How do the authors prevent contamination to avoid false positive results? Results: Authors should include normalization of read counts (e.g., per million mapped reads) in the results to improve comparability across different runs. Discussion: The authors should include considerations of the time and cost of the workflow to provide a sense of the feasibility of the method in clinics. ****** 6. 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: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0264855.r001
Revision 1
4 Jan 2022 Author Response Dear Reviewers, dear Editor, Thank you for considering and reviewing our manuscript. Please find below our response. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. AB: We have changed the style according to the PLOS ONE style templates and hope to meet all criteria. 2. Please review your reference list to ensure that it is complete and correct. AB: There have been no final changes to the reference list. 3. Please provide additional details regarding participant consent. We have added the Ethics statement to the manuscript (line 145-148) 4. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. We have added the Ethics statement to the manuscript (line 145-148) Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ________________________________________ 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: N/A ________________________________________ 3. Have the authors made all data underlying the findings in their manuscript fully available? Reviewer #1: Yes Reviewer #2: No AB: We have uploaded all data (PRJEB49379 at the European Nucleotide Archive) ________________________________________ 4. Is the manuscript 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 note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ________________________________________ 5. Review Comments to the Author Reviewer #1: The authors describe a method based on the combination of multiplex PCR with MinION sequencing for the simultaneous detection of SARS-CoV-2 and a panel of potential co-infecting agents. The study was well conducted and no major issues were identified. Some minor comments are provided below. Please provide additional information on how the species identification was performed upon acquisition of the reads. AB: Dear Reviewer #1, thank you for reviewing our manuscript. Species identification was performed by generating reference alignments with subsequent blast for validation. We have included additional information (line 136-137). Use the term "Cq" instead of "Ct" according to Bustin et al., throughout the manuscript, including in the tables. AB: We have changed this accordingly. In line 161 a "." seems to be missing in the Cq value. AB: We have corrected this (should have been a “-“) Whenever referring to Tables and Figures, start with with capital "T" and/ or "F". Tables should be improved, provide captions with additional information. AB: We have corrected this and included additional information in the captions of Table 2 and 3. With the data obtained can the authors provide details regarding the analytical sensitivity, specificity and accuracy of their method? AB: We have included some general considerations in the discussion (line 284 – 290) Reviewer #2: Brinkmann and colleagues presented and evaluated an alternative method for simultaneous identification of respiratory pathogens using amplicon sequencing on a nanopore platform. The method was evaluated with clinical samples and compared with commercially available multiplex screening assays. In addition, the methodology was tested in an international quality assurance exercise. The study is interesting and valuable because respiratory pathogens other than SARS-CoV-2 are often undiagnosed during the pandemic. Sequencing using the Nanopore platform is also becoming more available worldwide. The manuscript is concise and clearly written, although some minor improvements should be made. Title: the proposed panel also is intended to detect some bacterial pathogens. It would be good to adjust the title accordingly to indicate the broader scope of this method. AB: Dear Reviewer #2, thank you for your suggestions. We have added “bacteria” To the title. Data access statement: the authors state that all data are available through the SRA databases. The accession number should be included in the manuscript. AB: We have uploaded all data (PRJEB49379 at the European Nucleotide Archive) and included the data access statement in the methods section. Bioinformatics analysis: the author used the standard software provided by the sequencing company, which could be advantageous in a clinical setting, but the authors may wish to compare their results with more advanced workflows available for nanopore reads. AB: Although comparison of analysis workflows might be interesting, we think that this is not scope of the manuscript. As only short target amplicons need to be evaluated, aligning the reads to reference targets is the most simple and straightforward approach without the need for benchmarking. Please provide further details on the primer trimming method. AB: Added to the bioinformatics section. How do the authors prevent contamination to avoid false positive results? AB: All results are evaluated based on the negative control in the run (line 137-141) to monitor contamination. To avoid contaminations in the lab, we premise general precautions. However, theses are not specifically addressed in the manuscript. Results: Authors should include normalization of read counts (e.g., per million mapped reads) in the results to improve comparability across different runs. AB: For comparability, we included the ratio of target reads / total reads in the sample (table 2, table 4). Discussion: The authors should include considerations of the time and cost of the workflow to provide a sense of the feasibility of the method in clinics. AB: We have included some general considerations in the discussion (line 284 – 290) ________________________________________ Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0264855.r002
18 Feb 2022 Decision Letter - Kok Keng Tee, Editor RespiCoV: Simultaneous identification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and 46 respiratory tract viruses and bacteria by amplicon-based Oxford-Nanopore MinION sequencing PONE-D-21-32371R1 Dear Dr. Brinkmann, 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, Kok Keng Tee, Ph.D. Section Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) Reviewer #2: N/A ****** 4. Have the authors made all data underlying the findings in their manuscript 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 manuscript 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 note any specific errors here. 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: All the comments have been addressed and no further information is required. From this reviewer's point of view the manuscript is acceptable for publication. Reviewer #2: Brinkmann and colleaques present a revised manuscript where all comments have been adressed carefully by the authors. ****** 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:** Alejandro Garrido-Maestu Reviewer #2: No https://doi.org/10.1371/journal.pone.0264855.r003
Formally Accepted
28 Feb 2022 Acceptance Letter - Kok Keng Tee, Editor PONE-D-21-32371R1 RespiCoV: Simultaneous identification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and 46 respiratory tract viruses and bacteria by amplicon-based Oxford-Nanopore MinION sequencing Dear Dr. Brinkmann: 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. Kok Keng Tee Section Editor PLOS ONE https://doi.org/10.1371/journal.pone.0264855.r004

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