Development of an efficient Sanger sequencing-based assay for detecting SARS-CoV-2 spike mutations

Ho Jae Lim; Min Young Park; Hye Soo Jung; Youngjin Kwon; Inhee Kim; Dong Kwan Kim; Nae Yu; Nackmoon Sung; Sun-Hwa Lee; Jung Eun Park; Yong-Jin Yang

doi:10.1371/journal.pone.0260850

Peer Review History

Original SubmissionAugust 6, 2021
20 Sep 2021 Decision Letter - Baochuan Lin, Editor PONE-D-21-25453Development of efficient Sanger sequencing-based assay system for SARS-CoV-2 spike variantsPLOS ONE Dear Dr. Yang, 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. I have received the reviews of your manuscript. While your paper addresses an interesting question, the reviewers stated several concerns about your study and did not recommend publication in its present form. The presentation as well as the readability of the manuscript need to be improved, since the manuscript as a whole is quite convoluted. The title of the manuscript implied that the PCR plus Sanger Sequencing provided the necessary information. However, this is not clearly conveyed in the main text. The abstract only mentioned the 5-SSG primer assay development. From the main text, the 5-SSG primer assay system does not include sequence, how do the authors determine the mutation detected, PCR product size? In addition, the quality of the language needs to be improved, there are quite a few awkward sentences and typos throughout the manuscript. Please have a fluent, preferably native, English-language speaker thoroughly copyedit your manuscript for language usage, spelling, and grammar. Specific comments: Need to declare that Jeong-Eui Lee is the Seegene employee Line 88 – 91, please rephrase for clarity. What really did the authors develop? PCR assay in conjunction with Sanger sequencing or just PCR assay? Line 99, “redesigned or modified” from what? Line 117 – 120, awkward sentence, please rephrase for clarity. Also, are these viruses, bacteria and fungi selected causing respiratory infections? What is the selection criteria? Line 119, suggest changing “2 other kinds of coronaviruses” to “Coronavirus OC43 & 229E” Line 135 – 137, awkward sentences, please rephrase for clarity. Line 146 – 147, this sentence is confusing, please rephrase. Line 198 – 199, please rephrase for clarity. Please submit your revised manuscript by Nov 04 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. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Baochuan Lin, Ph.D. Academic Editor PLOS ONE Journal requirements: When submitting your revision, we need you to address these additional requirements. 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 2. 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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. [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: 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: No Reviewer #2: 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 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: Lim et al. have developed SARS-CoV-2 Spike protein primer sets to distinguish VOC and VOI as defined by WHO. The primers seem to work well for the VOC strains and some of the VOI strains. However, recently, lambda (June 14) and mu (August 30) have been newly designated. Since mu is designated after the submission date of August 6, authors can dismiss the VOI. The lambda (C.37) has 246_253delinsN deletion, which coincides with the reverse primer of 144S. Authors need to check whether the current primer design can detect the deletion in the lambda variant. Also, authors needs to share the raw data of the sequencing data prior to the publication. Reviewer #2: This study describes development and testing of five primer sets for detecting SARS-CoV-2 spike variants using Sanger sequencing. The manuscript is well written, and was a pleasure to read. The study appears robust, apt, and timely, especially considering its potential for application in clinical settings. I only have a few minor comments: 1. A few typos: -line 38, 'detect nine', instead of 'detect of nine' -line 43, 'ability of the 5-SSG primer', instead of 'ability of 5-SSG primer'. Same for lines 250, 273, 284. -line 81, 'methods', instead of 'method' 2. In the first paragraph of the results section (lines 204-214) and throughout the manuscript, the authors refer to 'mutants' as 'variants'. The term 'variant' in SARS-CoV-2 literature is probably more appropriately used for a constellation of mutations that make up a genetically (and usually epidemiologically) distint virus, rather than single mutations, and I fear that the authors use of the word here may not be appropriate. 3. Under acknowledgement, the name of the person being 'thanked' is missing. ****** 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: Yes:** Takahiko Koyama 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.0260850.r001
Revision 1
13 Oct 2021 Author Response Responses to Academic Editor Comments 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. I have received the reviews of your manuscript. While your paper addresses an interesting question, the reviewers stated several concerns about your study and did not recommend publication in its present form. The presentation as well as the readability of the manuscript need to be improved, since the manuscript as a whole is quite convoluted. The title of the manuscript implied that the PCR plus Sanger Sequencing provided the necessary information. However, this is not clearly conveyed in the main text. The abstract only mentioned the 5-SSG primer assay development. From the main text, the 5-SSG primer assay system does not include sequence, how do the authors determine the mutation detected, PCR product size? In addition, the quality of the language needs to be improved, there are quite a few awkward sentences and typos throughout the manuscript. Please have a fluent, preferably native, English-language speaker thoroughly copyedit your manuscript for language usage, spelling, and grammar. A: Thank you for your valuable comment. To improve the quality of the manuscript's expression and readability, we requested a native-speaker proofreading service, and the revised manuscript has been resubmitted here. Also, as per your suggestion, the title has been changed to “Development of an efficient Sanger sequencing-based assay for detecting SARS-CoV-2 spike mutations", to more accurately include the essential information provided by PCR and Sanger sequencing. All additional corrections made in the manuscript are marked in red. Electrophoresis was performed to determine the size of the PCR product, and the results are summarized in Tables 2 and S2. For reference, photos of the electrophoresis have been attached below. Specific comments: 1. Need to declare that Jeong-Eui Lee is the Seegene employee A: We have carefully reconsidered the issue of Jeong-Eui Lee as a co-author. Since his direct contribution to the paper is not entirely clear, we have decided to remove his authorship. We apologize for any confusion we may have caused by not initially making a more careful decision. 2. Line 88 – 91, please rephrase for clarity. What really did the authors develop? PCR assay in conjunction with Sanger sequencing or just PCR assay? A: We have modified the manuscript’s passage to say, “PCR assay in conjunction with Sanger sequencing”, and marked these changes in red. 3. Line 99, “redesigned or modified” from what? A: We have modified the sentence passage to state, ““modified from Global Initiative on Sharing Avian Influenza Data (GISAID) database, with a frequency cut-off > 1%, applied with degenerative or inosine to optimize melting temperature (Tm), avoid repetitive sequences, and include GC content > 65%, using Gene Runner (ver. 6.0) [20, 21].”, and marked these changes in red. 4. e 117 – 120, awkward sentence, please rephrase for clarity. Also, are these viruses, bacteria and fungi selected causing respiratory infections? What is the selection criteria? A: We have chosen here to focus on all pathogen species for which standard stains could be obtained to compare analytical capabilities. Among them, the results were separated between those causing respiratory infection (Table 2), and other pathogens (Supplementary Table 2) for clarity. 5. Line 119, suggest changing “2 other kinds of coronaviruses” to “Coronavirus OC43 & 229E” A: As per your suggestion, we have changed the sentence to “Coronavirus OC43 & 229E”, and marked the revision in red. 6. Line 135 – 137, awkward sentences, please rephrase for clarity. A: As follow your suggestion, we have reworded the sentences for clarity, and marked these changes in red. 7. Line 146 – 147, this sentence is confusing, please rephrase. A: The sentence has been rephrased, and is marked in red. 8. Line 198 – 199, please rephrase for clarity. A: The passage has been rephrased, and is marked in red. Responses to Reviewer Comments Reviewer #1’s comments and responses ▶ Comment 1: Lim et al. have developed SARS-CoV-2 Spike protein primer sets to distinguish VOC and VOI as defined by WHO. The primers seem to work well for the VOC strains and some of the VOI strains. However, recently, lambda (June 14) and mu (August 30) have been newly designated. Since mu is designated after the submission date of August 6, authors can dismiss the VOI. The lambda (C.37) has 246_253delinsN deletion, which coincides with the reverse primer of 144S. Authors need to check whether the current primer design can detect the deletion in the lambda variant. Also, authors needs to share the raw data of the sequencing data prior to the publication. ▶ Response to comment 1: Thank you for your very important comments. We have checked whether the 5-SSG primer assay system can detect deletion in Mu and lambda variants. As you point out, the lambda (C.37) 246_253delinsN deletion is not amplified under the influence of 144S reverse primer, but it can be confirmed with the 417S primer set. In addition, there is no problem in confirming Mu (B. 1.621) mutations. We have attached the figure below to support this information. Since your comments are very important, so we will monitor the occurrence of mutations and continue to improve our primer sets. As your request, we have included a new supplementary figure of raw data for the sequencing results of the key mutants, and have enclosed a data file to share the NGS raw data in FASTA format. Reviewer #2’s comments and responses This study describes development and testing of five primer sets for detecting SARS-CoV-2 spike variants using Sanger sequencing. The manuscript is well written, and was a pleasure to read. The study appears robust, apt, and timely, especially considering its potential for application in clinical settings. I only have a few minor comments: ▶ Comment 1: A few typos: - line 38, 'detect nine', instead of 'detect of nine' - line 43, 'ability of the 5-SSG primer', instead of 'ability of 5-SSG primer'. Same for lines 250, 273, 284 - line 81, 'methods', instead of 'method' be appropriate. ▶ Response to comment 1: All errors in manuscript have been corrected, and are marked in red. ▶ Comment 2: In the first paragraph of the results section (lines 204-214) and throughout the manuscript, the authors refer to 'mutants' as 'variants'. The term 'variant' in SARS-CoV-2 literature is probably more appropriately used for a constellation of mutations that make up a genetically (and usually epidemiologically) distint virus, rather than single mutations, and I fear that the authors use of the word here may not be appropriate. ▶ Response to comment 2: We agree with your valuable comment. The inappropriate words in manuscript have been corrected and are marked in red. ▶ Comment 3: Under acknowledgement, the name of the person being 'thanked' is missing. ▶ Response to comment 3: We have been added the name of Editage in acknowledgement and marked it in red. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0260850.r002
10 Nov 2021 Decision Letter - Baochuan Lin, Editor PONE-D-21-25453R1Development of an efficient Sanger sequencing-based assay for detecting SARS-CoV-2 spike mutationsPLOS ONE Dear Dr. Yang, 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. Both reviewers agreed that the revised version has addressed most of the comments raised and showed significant improvement. However, one of the reviewers still feel that one significant point regarding delta variant needs to be addressed. In addition, I also have a few points that still need to be addressed (see specific comments below). Specific comments:1. Line 135 - 136, "Anonymized residual of 17 nasopharyngeal swab specimens SARS-CoV-2 positive..." suggest changing to "Anonymized residual of 17 SARS-CoV-2 positive nasopharyngeal swab specimens..."2. Line 146 - 147, "The comparative LOD assessment was performed 25 times until the concentration of the targeted band was detected as positive." Not sure what the authors want to convey, please rephrase for clarity.3. Line 150 & 152, change PCR to RT-PCR. Please submit your revised manuscript by Dec 25 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. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Baochuan Lin, Ph.D. Academic Editor PLOS ONE Journal Requirements: 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. [Note: HTML markup is below. Please do not edit.] 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: N/A Reviewer #2: (No Response) ****** 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: Authors have responded to my review comments on 246_253delinsN on lambda variant. However, since this is an important point, authors should address that their assay works for the lambda and others in discussion. Reviewer #2: Thank you for addressing my previous comments and others'. I think the current version of the manusript is much improved. ****** 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:** Takahiko Koyama 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.0260850.r003
Revision 2
15 Nov 2021 Author Response Specific comments 1. Line 135 - 136, "Anonymized residual of 17 nasopharyngeal swab specimens SARS-CoV-2 positive..." suggest changing to "Anonymized residual of 17 SARS-CoV-2 positive nasopharyngeal swab specimens..." A: Thank you for your valuable comment. We have modified this text in the manuscript as “anonymized residual of 17 SARS-CoV-2 positive nasopharyngeal swab specimens of patients diagnosed with SARS-CoV-2 positive between February and June 2021 were obtained and used for this study” (lines 145-148). 2. Line 146 - 147, "The comparative LOD assessment was performed 25 times until the concentration of the targeted band was detected as positive." Not sure what the authors want to convey, please rephrase for clarity. A: We have modified the sentence as follows for clarity: “For the performance analysis of 5-SSG primers, 25 replicates were performed. The comparative LOD was determined as the minimum detectable concentration” (lines 157-159). 3. Line 150 & 152, change PCR to RT-PCR. A: As per your suggestion, we have changed the subheading and sentence to RT-PCR (lines 162, 164). Responses to Reviewer Comments Reviewer #1’s comments and responses ▶ Comment: Authors have responded to my review comments on 246_253delinsN on lambda variant. However, since this is an important point, authors should address that their assay works for the lambda and others in discussion. ▶ Response to comment: Thank you for your valuable comment. As suggested, we mentioned the lambda variant issue in this study, as the 246_253delinsN mutation, in the discussion section of the manuscript (lines 337-341). Attachments Attachment Submitted filename: Response to reviewer.docx https://doi.org/10.1371/journal.pone.0260850.r004
18 Nov 2021 Decision Letter - Baochuan Lin, Editor Development of an efficient Sanger sequencing-based assay for detecting SARS-CoV-2 spike mutations PONE-D-21-25453R2 Dear Dr. Yang, 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, Baochuan Lin, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0260850.r005
Formally Accepted
6 Dec 2021 Acceptance Letter - Baochuan Lin, Editor PONE-D-21-25453R2 Development of an efficient Sanger sequencing-based assay for detecting SARS-CoV-2 spike mutations Dear Dr. Yang: 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. Baochuan Lin Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0260850.r006

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