Whole genome sequencing of SARS-CoV2 strains circulating in Iran during five waves of pandemic

Jila Yavarian; Ahmad Nejati; Vahid Salimi; Nazanin Zahra Shafiei Jandaghi; Kaveh Sadeghi; Adel Abedi; Ali Sharifi Zarchi; Mohammad Mehdi Gouya; Talat Mokhtari-Azad

doi:10.1371/journal.pone.0267847

Peer Review History

Original SubmissionDecember 14, 2021
20 Jan 2022 Decision Letter - Etsuro Ito, Editor PONE-D-21-38222Whole genome sequencing of SARS-CoV2 strains circulating in Iran during five waves of pandemicPLOS ONE Dear Dr. Mokhtari-Azad, 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. The language should be improved. Please submit your revised manuscript by Mar 06 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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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: "Authors would like to express their great thanks to all data contributors, i.e. the authors and their originating laboratories responsible for obtaining the specimens, and their submitting laboratories for generating the genetic sequence and metadata and sharing via the GISAID Initiative, on which this research is based. We thank GISAID for all their support. A part of this study is supported by NIMAD under grant number 994376." 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: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." 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 financial disclosure: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." At this time, please address the following queries: a) Please clarify the sources of funding (financial or material support) for your study. 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To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ 5. 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. 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 Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: Yes Reviewer #3: 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: Yes Reviewer #3: 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 Reviewer #3: 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: Regarding the manuscript entitled “Whole genome sequencing of SARS-CoV2 strains circulating in Iran during five waves of pandemic”: The authors performed an extensive analysis of the SARS-CoV-2 genomes in Iran to find information about the SARS-CoV2 lineages, variants of interests and variants of concerns during five waves of pandemic. Overall it is a well-organized manuscript and a nice contribution to the field and the information presented in this manuscript can be beneficial to the other researchers. Below are some suggestions that the authors may wish to consider to improve their manuscript: -Please mention to the COVID-19 in the introduction section. -Please mention to the different known clades of SARS-CoV-2 in the introduction section. -In the figure legend please replace “constricted” by “constructed” -The number of viruses in the phylogenetic tree is 53. -Since the purpose of the manuscript was finding information about the viral lineages, variants of interests and variants of concern, it would be suggested to describe them briefly. -Please add the following reference “Usage of peptidases by SARS-CoV-2 and several human coronaviruses as receptors: A mysterious story” in the line 366. Reviewer #2: The manuscript presents valuable information about SARS-CoV2 viruses circulating in Iran during 5 waves of the pandemic. It is well written and organized. Only few revisions are suggested: - The Introduction mostly explains the virus proteins and structure, while it might be preferred that first the Introduction part talks also about why this virus is important why this study was designed. - It is suggested that the mutations in different proteins (non-structural, structural and accessory) are entered and organized in a table, so the reader can easily see and follow different mutations in different proteins during different waves of the pandemic in Iran. - Line 486: … which the results were compatible with … -- needs to be revised to: … of which the results were compatible with … Reviewer #3: This is a thorough analysis of the genome sequences of SARS-CoV-2 viruses in circulation in Iran over five waves of the pandemic. The full genome sequence of five to twenty viruses (10,10, 9, 5 and 20) from each wave were analysed by next generation sequencing and the results analysed to report here ion what variation has been seen. The data quality has not been described fully: for example, how complete each genome sequence turned out to be and what proportion of undetermined nucleotides were in the genome sequence assembly. This would provide some kind of quality assessment of the gene sequence data. The gene sequence data have been shared in the EpiCov database of GISAID, but I have not checked the sequences fully. The catalogue of changes seen and described in the results is comprehensive, but going through the results for each virus polypeptide is demanding for the reader and I wondered if the authors could come up with some kind of graphical display to supplement the description of what was seen and elaborated on in the text. I think for most virus polypeptides this could be relatively easily done, but, like in spike (for which there is a dedicated table), the variation in NSP3 seems quite extensive. Nevertheless, I think a graphical representation would enhance the message of the manuscript greatly. In the discussion the authors summarise a lot of literature on the likely effects of amino acid substitutions. My feeling is that the authors should differentiate between results and conclusions made by experiment from those that have been generated by modelling. Moreover, there is frequent use of phrases like ‘viral oligomerisation interface’ (e.g. lines 136, 140, 141, 147, 150, 151 etc.). Here I was not clear in many cases what the evidence for this conclusion was. I might have missed the references to many of these. In addition, it was not what was meant precisely by a ‘viral oligomerisation interface’. I was not sure of this meant oligomerisation of the relevant polypeptide or something else. There are also two places in which antibody recognition sites are described for the NSP7 and NSP8 (lines 175 and 179). Do the authors know if this recognition is by post-infection serum or something else? Also, references should be given for this. NSP7 was also stated to undergo antigenic drift. I wonder if this is better defined as antigenic change, or change in a T-cell epitope. This is also referred to on line 314. The phylogenetic tree shown in the figure uses the GISAID clade nomenclature. I wonder if it is possible to correlate these clades and subclades with the PANGO lineages. I think it might also be useful to include a graph of the five waves of COVID-19 in Iran and indicate when the samples were taken on the graph. Minor points. In many cases the authors refer to mutations in the virus polypeptides. I think, strictly speaking, mutations occur in genes that encode mutant proteins that have amino acid substitutions. I would recommend that this is considered by the authors. There are a number of places where the English is not correct – the indefinite and the definite articles are missing in some number of places. This should be corrected by asking for additional help from colleagues. Some other points include: Generally, use family names only when referring to work done by others. Also, the word ‘data’ has been used both as a singular term (incorrectly) and as a plural (correctly). There should be consistency. I would suggest that when referring to a series of amino acid substitutions the order of these substitutions is always from the N-terminal to C-terminal of the polypeptide. Line 62, define what a ‘primase’ is. Line 69. I suggest saying that Spike ‘mediates’ attachment and entry rather than is ‘important for’ attachment and entry. Line 88. I think ‘designated’ is not the right word here – perhaps ‘selected’. Line 98. I suggest ‘Library construction was done by using…’ Line 99. Explain what ‘captured’ means in this context. Line 236. Define in which wave hCoV-19/Iran/Gilan/NICS1-58/2020 was isolated. Line 295. The word ‘third’ should be ‘thirds’. Line 305. “Recommended’ seems to be the wrong word here. Line 346. Add the words ‘change in’ ahead of ‘incubation period’. Line 366. I think this should be Receptor Binding Domain (not Receptor Binding Motif). Lines 455 to 456. I am not clear what intraviral connections refers to. Lines 453 to 457 might be better composed. Line 481. The authors comment on the ‘rate’ of mutation. However, mutations rates have not been addressed in this work. This needs re-wording to reflect more accurately what has been observed in this study. ****** 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 Reviewer #3: No 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.0267847.r001
Revision 1
13 Feb 2022 Author Response Dear Editor-in-Chief of PLOS ONE, Thank you very much for the review of our manuscript entitled: “Whole genome sequencing of SARS-CoV2 strains circulating in Iran during five waves of pandemic”. We sincerely appreciate all valuable comments and suggestions, which helped us to improve the quality of the article. Our responses to the Reviewers’ comments are described below in a point-to-point manner. Appropriated changes, suggested by the Reviewers, have been introduced to the manuscript (highlighted within the document). The language is improved. Dear Reviewer 1: The changes were highlighted in yellow. - Please mention to the COVID-19 in the introduction section. The sentence was added to the beginning of the Introduction. - Please mention to the different known clades of SARS-CoV-2 in the introduction section. Was added to the last paragraph of Introduction. - In the figure legend please replace “constricted” by “constructed” Done. - The number of viruses in the phylogenetic tree is 53. Phylogenetic tree was corrected. - Since the purpose of the manuscript was finding information about the viral lineages, variants of interests and variants of concern, it would be suggested to describe them briefly. Was described at the end of Introduction. - Please add the following reference “Usage of peptidases by SARS-CoV-2 and several human coronaviruses as receptors: A mysterious story” in the line 366. Was added. Dear Reviewer 2: The changes were underlined. - The Introduction mostly explains the virus proteins and structure, while it might be preferred that first the Introduction part talks also about why this virus is important why this study was designed. It was added to the end of Introduction. - It is suggested that the mutations in different proteins (non-structural, structural and accessory) are entered and organized in a table, so the reader can easily see and follow different mutations in different proteins during different waves of the pandemic in Iran. Mutations of nonstructural genes illustrated in the figure. - Line 486: … which the results were compatible with … -- needs to be revised to: … of which the results were compatible with … Corrected. Dear Reviewer 3: The changes were highlighted in green. This is a thorough analysis of the genome sequences of SARS-CoV-2 viruses in circulation in Iran over five waves of the pandemic. The full genome sequence of five to twenty viruses (10,10, 9, 5 and 20) from each wave were analyzed by next generation sequencing and the results analyzed to report here on what variation has been seen. - The data quality has not been described fully: for example, how complete each genome sequence turned out to be and what proportion of undetermined nucleotides were in the genome sequence assembly. This would provide some kind of quality assessment of the gene sequence data. The gene sequence data have been shared in the EpiCov database of GISAID, but I have not checked the sequences fully. Added to the materials and methods section. - The catalogue of changes seen and described in the results is comprehensive, but going through the results for each virus polypeptide is demanding for the reader and I wondered if the authors could come up with some kind of graphical display to supplement the description of what was seen and elaborated on in the text. I think for most virus polypeptides this could be relatively easily done, but, like in spike (for which there is a dedicated table), the variation in NSP3 seems quite extensive. Nevertheless, I think a graphical representation would enhance the message of the manuscript greatly. Mutations of nonstructural genes illustrated in the figure. - In the discussion the authors summarize a lot of literature on the likely effects of amino acid substitutions. My feeling is that the authors should differentiate between results and conclusions made by experiment from those that have been generated by modelling. Moreover, there is frequent use of phrases like ‘viral oligomerisation interface’ (e.g. lines 136, 140, 141, 147, 150, 151 etc.). Here I was not clear in many cases what the evidence for this conclusion was. I might have missed the references to many of these. In addition, it was not what was meant precisely by a ‘viral oligomerisation interface’. I was not sure of this meant oligomerisation of the relevant polypeptide or something else. That is really important point. There is not any reference about the role of some proteins as viral oligomerization interfaces, we added this, just extracted from GISAID mutation analysis, then we deleted them. - There are also two places in which antibody recognition sites are described for the NSP7 and NSP8 (lines 175 and 179). Do the authors know if this recognition is by post-infection serum or something else? Also, references should be given for this. NSP7 was also stated to undergo antigenic drift. I wonder if this is better defined as antigenic change, or change in a T-cell epitope. This is also referred to on line 314. It is another important point which is written just according to GISAID mutation analysis and there is not publication or in vitro studies, then we removed those from the article. - The phylogenetic tree shown in the figure uses the GISAID clade nomenclature. I wonder if it is possible to correlate these clades and subclades with the PANGO lineages. We could change the clades to PANGO in the tree, but because we used mostly clades in the text, then we prefer to put clades, if it is necessary we could change them. - I think it might also be useful to include a graph of the five waves of COVID-19 in Iran and indicate when the samples were taken on the graph. It is useful to include a graph, but because of the article length and number of tables and figures, we just wrote the date of each wave and the number of assessed samples in each wave. Minor points. - In many cases the authors refer to mutations in the virus polypeptides. I think, strictly speaking, mutations occur in genes that encode mutant proteins that have amino acid substitutions. I would recommend that this is considered by the authors. All were corrected. - There are a number of places where the English is not correct – the indefinite and the definite articles are missing in some number of places. This should be corrected by asking for additional help from colleagues. Reviewed and corrected as much as possible. Some other points include: - Generally, use family names only when referring to work done by others. All were corrected. - Also, the word ‘data’ has been used both as a singular term (incorrectly) and as a plural (correctly). There should be consistency. Corrected. I would suggest that when referring to a series of amino acid substitutions the order of these substitutions is always from the N-terminal to C-terminal of the polypeptide. Corrected - Line 62, define what a ‘primase’ is. Added. - Line 69. I suggest saying that Spike ‘mediates’ attachment and entry rather than is ‘important for’ attachment and entry. Corrected. - Line 88. I think ‘designated’ is not the right word here – perhaps ‘selected’. Corrected. - Line 98. I suggest ‘Library construction was done by using…’ Corrected. - Line 99. Explain what ‘captured’ means in this context. The sentence was changed to clear explanation. - Line 236. Define in which wave hCoV-19/Iran/Gilan/NICS1-58/2020 was isolated. Cleared. - Line 295. The word ‘third’ should be ‘thirds’. Corrected. - Line 305. “Recommended’ seems to be the wrong word here. Corrected. - Line 346. Add the words ‘change in’ ahead of ‘incubation period’. Added. - Line 366. I think this should be Receptor Binding Domain (not Receptor Binding Motif). Corrected. - Lines 455 to 456. I am not clear what intraviral connections refers to. Lines 453 to 457 might be better composed. Corrected. - Line 481. The authors comment on the ‘rate’ of mutation. However, mutations rates have not been addressed in this work. This needs re-wording to reflect more accurately what has been observed in this study. It corrected. We mean the number of mutations. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0267847.r002
18 Mar 2022 Decision Letter - Etsuro Ito, Editor PONE-D-21-38222R1Whole genome sequencing of SARS-CoV2 strains circulating in Iran during five waves of pandemicPLOS ONE Dear Dr. Mokhtari-Azad, 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 fined attached.This file show the suggestions by one of the reviewers. Please submit your revised manuscript by May 02 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. 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, Etsuro Ito 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. 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 #3: 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 #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #3: Yes ****** 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 #3: 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 #3: No ****** 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: (No Response) Reviewer #3: The manuscript by Yavarian et al. has addressed many of the points I raised in my report. However, I think that there are still various sections of the manuscript where improvements to the flow or meaning can be made. I will attach a scan of a handwritten marked-up document for the authors to consider. However, there are places that the authors still need to address. The main places where the authors need to further modify the text are as follows. (Minor changes suggested are in the marked-up copy). Lines 108 to 110. The term ‘mutations’ remain in this section to be used for amino acid substitutions. The same is also seen in lines 113 and 116. I suggest the authors re-check for the appropriate usage. I have made some suggestions in the marked-up scanned copy. Line 140. Introduce the concept here of amino acid deletions e.g. using the phrase ‘a deletion of G66 (G66del) and S76 (S67del)…’ See also lines 162 to 164, and line 240 Lines 144 and 145. Introduce the concept of stop codons in NS7b and NS8 - and the effect of the stop codons on what can be expected to be translated. Line 171 is not clear what the authors are trying to say here. Perhaps it is that amino acid substitutions in NS3 deserve greater study in vitro. Lines 195 to 197. I have made suggestions for changes here in the marked copy. Line 211. It was not clear what the authors meant by ‘missense mutations’. Line 243. A reference is needed. Line 271. It was unclear what therapeutic effects were envisaged here. Line 276. Here the authors are presumably referring to amino acid substitutions in the nucleoprotein, not in the gene. Line 277. A reference is needed. Line 287. A reference is needed. Lines 289 to 291. It is not clear what the authors are saying that termination of the NS8 gene somehow affects spike affinity for the receptor. The authors should elaborate on this. ****** 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: No Reviewer #3: No 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. Attachments Attachment Submitted filename: Yavarian markup.pdf https://doi.org/10.1371/journal.pone.0267847.r003
Revision 2
13 Apr 2022 Author Response Dear Dear Editor-in-Chief of PLOS ONE, Thank you very much for the review of our manuscript entitled: “Whole genome sequencing of SARS-CoV2 strains circulating in Iran during five waves of pandemic”. We sincerely appreciate all valuable comments and suggestions, especially dear reviewer 3, which helped us to improve the quality of the article. Our responses to the Reviewers’ comments are described below in a point-to-point manner. Appropriated changes, suggested by the Reviewers, have been introduced to the manuscript. The language is improved by all corrections made by reviewer 3. Reviewer #3: The manuscript by Yavarian et al. has addressed many of the points I raised in my report. However, I think that there are still various sections of the manuscript where improvements to the flow or meaning can be made. I will attach a scan of a handwritten marked-up document for the authors to consider. However, there are places that the authors still need to address. The main places where the authors need to further modify the text are as follows. (Minor changes suggested are in the marked-up copy). Lines 108 to 110. The term ‘mutations’ remain in this section to be used for amino acid substitutions. The same is also seen in lines 113 and 116. I suggest the authors re-check for the appropriate usage. I have made some suggestions in the marked-up scanned copy. They all corrected. Line 140. Introduce the concept here of amino acid deletions e.g. using the phrase ‘a deletion of G66 (G66del) and S76 (S67del)…’ See also lines 162 to 164, and line 240. They all corrected. Lines 144 and 145. Introduce the concept of stop codons in NS7b and NS8 - and the effect of the stop codons on what can be expected to be translated. We added that stop codons lead to truncated proteins. Line 171 is not clear what the authors are trying to say here. Perhaps it is that amino acid substitutions in NS3 deserve greater study in vitro. It was re-written. Lines 195 to 197. I have made suggestions for changes here in the marked copy. Correction was made. Line 211. It was not clear what the authors meant by ‘missense mutations’. We wrote change in amino acid. Line 243. A reference is needed. Added. Line 271. It was unclear what therapeutic effects were envisaged here. It was not appropriate sentence, we removed it. Line 276. Here the authors are presumably referring to amino acid substitutions in the nucleoprotein, not in the gene. Corrected. Line 277. A reference is needed. Added. Line 287. A reference is needed. Added. Lines 289 to 291. It is not clear what the authors are saying that termination of the NS8 gene somehow affects spike affinity for the receptor. The authors should elaborate on this. We re-wrote it. Attachments Attachment Submitted filename: Response to Reviewers2.docx https://doi.org/10.1371/journal.pone.0267847.r004
18 Apr 2022 Decision Letter - Etsuro Ito, Editor Whole genome sequencing of SARS-CoV2 strains circulating in Iran during five waves of pandemic PONE-D-21-38222R2 Dear Dr. Mokhtari-Azad, 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, Etsuro Ito Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0267847.r005
Formally Accepted
22 Apr 2022 Acceptance Letter - Etsuro Ito, Editor PONE-D-21-38222R2 Whole genome sequencing of SARS-CoV2 strains circulating in Iran during five waves of pandemic Dear Dr. Mokhtari-Azad: 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 Prof. Etsuro Ito Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0267847.r006

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