Nanopore metagenomic sequencing for detection and characterization of SARS-CoV-2 in clinical samples

Nick P. G. Gauthier; Cassidy Nelson; Michael B. Bonsall; Kerstin Locher; Marthe Charles; Clayton MacDonald; Mel Krajden; Samuel D. Chorlton; Amee R. Manges

doi:10.1371/journal.pone.0259712

Peer Review History

Original SubmissionSeptember 2, 2021
19 Oct 2021 Decision Letter - Ruslan Kalendar, Editor PONE-D-21-28544Nanopore Metagenomic Sequencing for Detection and Characterization of SARS-CoV-2 in Clinical SamplesPLOS ONE Dear Dr. Manges, 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 Dec 03 2021 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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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating the following in the Acknowledgments Section of your manuscript: [We would like to acknowledge the Berkeley Existential Risk Initiative for providing funding for this project. We would also like to acknowledge the British Columbia Public Health Laboratory and the Medical Microbiology Laboratory at Vancouver General Hospital for providing RT-PCR testing for a subset of our samples, as well as John Tyson from the BC Centre for Disease Control for technical support and troubleshooting.] We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: [CN received funding from the Berkeley Existential Risk Initiative to perform this research. The funding institution played no role in the design of this study. URL: https://existence.org/] Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 3. Thank you for stating the following in the Competing Interests section: [I have read the journal's policy and the authors of this manuscript have the following competing interests: SDC holds equity in BugSeq Bioinformatics Inc]. Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf. 4. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. 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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. 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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 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 ****** 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 ****** 5. Review Comments to the Author Reviewer #1: Summary: The authors present an interesting manuscript characterizing the utility of a metagenomic next-generation sequencing approach for detecting SARS-CoV-2 in clinical samples. They illustrate strong specificity and sensitivity of the assay in samples with higher viral loads (Ct < 30). The major contribution of the proposed method is the utility of the assembled genomes to distinguish SARS-CoV-2 variants as verified by Variant of Concern PCR. The manuscript presents findings of original research that would be of interest to the wider research community. However, there are some concerns regarding the reporting quality which if addressed can significantly strengthen the manuscript. The introduction can be expanded, specifically, the last paragraph discussing the stated goals of the manuscript. What do the authors mean by negative and positive controls? Are these sample status based on the RT-PCR? Why are there 11 negative controls (line 241) while only 5 negative RT-PCR tests? Also if there are two false positives, how is this not reflected in the results for the sample classification before adjusting for barcode crosstalk (Table 2)? Restate specificity for Ct > 30 as 30 to max Ct, which is 38? How does the nanopore SISPA based MGS compare to other MGS for SARS-CoV-2? Figure 4 in its current format is not adding sufficient value to the manuscript. Instead it could show the phylogeny of the 10 genomes with a small subset of samples derived from BC vs other provinces to clearly illustrate the point that the genomes cluster closer with samples from BC. This is a major result of the manuscript and can be illustrated more convincingly. There are some contradictory statements in the manuscript regarding the novelty of mNGS for SARS-CoV-2. In the introduction, the authors state that existing efforts have detected SARS-CoV-2 based on mNGS (line 73). However in discussion (line 447), they state that this study provides the first analysis of mNGS for characterizing SARS-CoV-2. Please clarify how this study compares with the prior work and offers improvement of existing methodology. “Our assay was able to distinguish between the Alpha and Gamma variants, which was not possible with a VOC PCR.” This is slightly misleading considering the VOC PCR is able to distinguish alpha and gamma variants, e.g. for P25 and P26 studies. ****** 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 [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.0259712.r001
Revision 1
22 Oct 2021 Author Response Academic Editor: 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf We have rigorously reviewed the PLOS ONE style requirements and believe that our manuscript aligns with the requirements. 2. Thank you for stating the following in the Acknowledgments Section of your manuscript: [We would like to acknowledge the Berkeley Existential Risk Initiative for providing funding for this project. We would also like to acknowledge the British Columbia Public Health Laboratory and the Medical Microbiology Laboratory at Vancouver General Hospital for providing RT-PCR testing for a subset of our samples, as well as John Tyson from the BC Centre for Disease Control for technical support and troubleshooting.] We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: [CN received funding from the Berkeley Existential Risk Initiative to perform this research. The funding institution played no role in the design of this study. URL: https://existence.org/] Please include your amended statements within your cover letter; we will change the online submission form on your behalf. We have amended the manuscript document to remove funding in our acknowledgements section. We have no further information to add to the funding statement. 3. Thank you for stating the following in the Competing Interests section: [I have read the journal's policy and the authors of this manuscript have the following competing interests: SDC holds equity in BugSeq Bioinformatics Inc]. Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf. We have updated our competing interests statement to include the statement that was included in our cover letter and have submitted this updated cover letter to the manuscript submission portal. We confirm that our competing interests align with PLOS ONE policies on sharing data and materials. 4. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. Data accession numbers are available within the manuscript and the data have been made freely available online. 5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Supporting information captions have been included at the very end of the manuscript file in accordance with the supporting information guidelines. In-text citations have been reviewed and no changes have been made. 6. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. We have reviewed the reference list to ensure it is complete and correct. No revisions have been made to the reference list. Reviewer #1: Summary: The authors present an interesting manuscript characterizing the utility of a metagenomic next-generation sequencing approach for detecting SARS-CoV-2 in clinical samples. They illustrate strong specificity and sensitivity of the assay in samples with higher viral loads (Ct < 30). The major contribution of the proposed method is the utility of the assembled genomes to distinguish SARS-CoV-2 variants as verified by Variant of Concern PCR. The manuscript presents findings of original research that would be of interest to the wider research community. However, there are some concerns regarding the reporting quality which if addressed can significantly strengthen the manuscript. The introduction can be expanded, specifically, the last paragraph discussing the stated goals of the manuscript. We have expanded the last paragraph of the introduction to more explicitly state the objectives and goals of our study, as well as the implications of these results to the field of diagnostic infectious diseases. What do the authors mean by negative and positive controls? Are these sample status based on the RT-PCR? Why are there 11 negative controls (line 241) while only 5 negative RT-PCR tests? For each multiplexed flowcell, we ran one negative (for each of the 11 sequencing runs) and one positive control (for the first 6 flowcells, after which we were confident in our ability to successfully detect SARS-CoV-2). Our positive control was extracted SARS-CoV-2 virions from cell culture to ensure that our assay was able to detect SARS-CoV-2 reliably. The negative control we used was a blank viral transport medium control (ie. extracted and randomly amplified from a tube of viral transport medium that was sterile), which was used to ensure there was no significant contamination of our reagents or barcode crossover during sequencing. We previously described our negative control in lines 175-177 of the manuscript file. Line 225 has been updated to include more detail about our positive control. The five negative RT-PCR tests represent testing for 5 negative clinical specimens. so if there are two false positives, how is this not reflected in the results for the sample classification before adjusting for barcode crosstalk (Table 2)? These two false-positive, negative controls were not reflected in table two as these did not represent clinical specimens, but rather blank controls that likely had SARS-CoV-2 reads assigned to them due to barcode crosstalk. Restate specificity for Ct > 30 as 30 to max Ct, which is 38? Both Tables 2 and 3 have been updated to reflect the range of Ct values. Ct 30-38.7. Ct 38.7 was the maximum Ct value for any of our samples. How does the nanopore SISPA based MGS compare to other MGS for SARS-CoV-2? Although SISPA and other MGS methods have been used for detection of SARS-CoV-2 both in nasopharyngeal swabs, as well as wastewater and fecal samples, these studies did not report test performance metrics such as sensitivity and specificity and often report solely SARS-CoV-2 reads per sample and a Ct value. Furthermore, these studies have not included data on assay controls, making comparisons or assessments of performance difficult. We suspect that mNGS protocols without an amplification step would have lower sensitivity than a random amplification-based approach such as SISPA. There is some evidence for this in Mostafa et al. (2020), where they report false negative mNGS results for samples exhibiting a Ct value as low as Ct 21.0. However, without accurate test performance data to compare these other mNGS methods to our own, we decided not to include any comparisons to other studies in our manuscript due to differences in test performance reporting. Figure 4 in its current format is not adding sufficient value to the manuscript. Instead it could show the phylogeny of the 10 genomes with a small subset of samples derived from BC vs other provinces to clearly illustrate the point that the genomes cluster closer with samples from BC. This is a major result of the manuscript and can be illustrated more convincingly. Figure 4 along with the figure legend has been updated to include a subset of the closest three samples related to our clinical samples (almost entirely BC samples), as well as 25 randomly sampled Canadian and 25 randomly sampled global samples. Based on these new samples, the conclusion that our samples cluster most closely with BC samples can more accurately be drawn. We believe changes to this figure more accurately highlights this result. There are some contradictory statements in the manuscript regarding the novelty of mNGS for SARS-CoV-2. In the introduction, the authors state that existing efforts have detected SARS-CoV-2 based on mNGS (line 73). However in discussion (line 447), they state that this study provides the first analysis of mNGS for characterizing SARS-CoV-2. Please clarify how this study compares with the prior work and offers improvement of existing methodology. Although other studies have used mNGS to detect SARS-CoV-2 (as stated in the introduction), our work represents the first study to both, assess the performance (ie. sensitivity and specificity), as well as characterize SARS-CoV-2 genomes through phylogenetic analysis. Two previous preprints have used nanopore-based SISPA mNGS to detect SARS-CoV2 (ref 13) or to assess within host SARS-CoV-2 diversity (ref 12), but neither of these studies quantified performance metrics for their assay or demonstrated the utility of this assay for phylogenetic analysis. We have updated the introduction (lines 109-111), as well as the discussion (lines 476-479) to be clearer about how our study represents an important contribution to the literature. “Our assay was able to distinguish between the Alpha and Gamma variants, which was not possible with a VOC PCR.” This is slightly misleading considering the VOC PCR is able to distinguish alpha and gamma variants, e.g. for P25 and P26 studies. We have rephrased this statement to clarify that it only applies to the VOC PCR being used at Vancouver General Hospital for surveillance in British Columbia. Attachments Attachment Submitted filename: PLoSONE_rebuttal_letter_FINAL.docx https://doi.org/10.1371/journal.pone.0259712.r002
26 Oct 2021 Decision Letter - Ruslan Kalendar, Editor Nanopore Metagenomic Sequencing for Detection and Characterization of SARS-CoV-2 in Clinical Samples PONE-D-21-28544R1 Dear Dr. Manges, 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, Ruslan Kalendar Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0259712.r003
Formally Accepted
5 Nov 2021 Acceptance Letter - Ruslan Kalendar, Editor PONE-D-21-28544R1 Nanopore Metagenomic Sequencing for Detection and Characterization of SARS-CoV-2 in Clinical Samples Dear Dr. Manges: 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 Professor Ruslan Kalendar Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0259712.r004

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