Origin of imported SARS-CoV-2 strains in The Gambia identified from whole genome sequences

Abdoulie Kanteh; Jarra Manneh; Sona Jabang; Mariama A. Kujabi; Bakary Sanyang; Mary A. Oboh; Abdoulie Bojang; Haruna S. Jallow; Davis Nwakanma; Ousman Secka; Anna Roca; Alfred Amambua-Ngwa; Martin Antonio; Ignatius Baldeh; Karen Forrest; Ahmadou Lamin Samateh; Umberto D’Alessandro; Abdul Karim Sesay

doi:10.1371/journal.pone.0241942

Peer Review History

Original SubmissionOctober 22, 2020
6 Jan 2021 Decision Letter - Yury E Khudyakov, Editor PONE-D-20-33232 Origin of imported SARS-CoV-2 strains in The Gambia identified from whole genome sequences PLOS ONE Dear Dr. Kanteh, 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. ============================== Your manuscript was reviewed by 4 experts in the field. They identified many important problems in your submission. Please carefully consider the attached comments and provide point-by-point responses. ============================== Please submit your revised manuscript by Feb 18 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Yury E Khudyakov, PhD 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. We note that you are reporting an analysis of a microarray, next-generation sequencing, or deep sequencing data set. PLOS requires that authors comply with field-specific standards for preparation, recording, and deposition of data in repositories appropriate to their field. Please upload these data to a stable, public repository (such as ArrayExpress, Gene Expression Omnibus (GEO), DNA Data Bank of Japan (DDBJ), NCBI GenBank, NCBI Sequence Read Archive, or EMBL Nucleotide Sequence Database (ENA)). In your revised cover letter, please provide the relevant accession numbers that may be used to access these data. For a full list of recommended repositories, see http://journals.plos.org/plosone/s/data-availability#loc-omics or http://journals.plos.org/plosone/s/data-availability#loc-sequencing 3. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (i) whether consent was informed and (ii) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information. If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information. Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”). For additional information about PLOS ONE ethical requirements for human subjects research, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research. 4. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. [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: Partly Reviewer #3: Partly Reviewer #4: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: N/A Reviewer #3: No Reviewer #4: 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: No Reviewer #4: 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 Reviewer #3: Yes Reviewer #4: 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: Dear author(s), I have read your submission and it was interesting. Only there are minor things that you should consider for having better insight to your study. You may pay attention to this point that whether or not this study has been done previously by others and compare your results. Another point that you should consider is that what is your main goal for future of performing this work and what was your expectation out of that data. Reviewer #2: In this paper, Kanteh et al. reported whole genome sequence of SARS-CoV-2 strains detected in Gambia and their phylogenetic analysis with other strains outside of the country. Given little viral sequence information from African countries, the study provides us with a clue to elucidate the global transmission dynamics of the virus at the relatively early time point of the current outbreak. However, I have several concerns in the manuscript to be addressed before the publication. Major points 1. Absolute “origin” or “source” cannot be inferred from phylogenetic tree. That is because there must be other viral strains from other countries that are more closely related to Gambian strains but unsampled and not included in their phylogenetic analysis. 2. Related to the point #1, viral sequences from other countries included in the phylogenetic analysis are very few. There should be many more available sequence data of viral strains collected by March 2020 from all over the world in GISAID, ViPR, and so on. 3. Difference in sequences between Illumina and Nanopre protocols should be done by not phylogenetic trees but direct comparison of SNPs (number and positions) between them. 4. Related to the point #3, the authors had better show either of only Figure 2 or Figure 3. Minor points 5. There are a lot of typo in the manuscript. Please read the manuscript carefully and correct them before re-submission. 6. (Lines 34 and 203) “Wuhan reference genome” is not a scientific term. Please indicate the actual strain name as described in the line 177. 7. Texts in the lines 76 and 77-78 are redundant. 8. The names of genes for the screening and confirmation should be specified here (as described in the Table 2.) 9. I personally think Figure 1 is unnecessary and can be removed from the manuscript. Yet, I will leave the decision to the editor and authors. 10. In the table 2, there is “Ct” in the third column title. However, I do not see any Ct values in the table. 11. (Line 249) WGS should be spelled out. Reviewer #3: The authors conducted complete genomic sequencing of the first 6 cases of SARS-CoV-2 in The Gambia to determine/confirm their origin/genetic characteristics using Illumina MiSeq and Nanopore platforms. The study addresses an important issue because the knowledge on the genetic characteristic of SARS-CoV-2 strains circulating in The Gambia/Adrica. The study also appears to be technically sound. The results presented/discussed are insufficient for a full-length research article. If no additional analysis (see below) is done, the submission must be converted to a short communication. Overall, the manuscript is too focused on technical details of both sequencing methods employed and presents very little detail on the genomic/molecular analysis of the Gambian SARS-CoV-2 strains. This has to be changed by mostly re-writing the manuscript. It is not clear why the two sequencing approaches had to be used: Illumina MiSeq and Nanopore. Although, the authors stated that both methods had their own advantages, it is not clear why the two methods were needed to just determine the strain origin. “While Illumina sequencing may be more accurate in determining within sample-diversity, Nanopore data can help with the understanding of the linkage between SNPs within individual virions”. The importance/relevance of determining within host/virion diversity in this study is not clear. It is well established fact that coronaviruses exist as quasispecies within the same host. The abstract needs to be re-written. Currently, it’s a mere statement of the methodology used in the study. It has to be re-written to properly convey the problem/background, approach, findings and conclusion of the study. Accession numbers for the newly generated sequences must be provided, but were missing. Issues with the analysis: More SARS-CoV-2 strains should be included in the phylogenetic trees. Phylogenetic distance and bootstrap values should be reflected on the trees. The meaning of the dashed lines should be explained in the figure legends. Recombination analysis should be conducted. Reviewer #4: This is a nice short paper on sequencing some SARS-CoV-2 genomes from Gambia. their methods seem fine, and the sequences seem reasonable. My only concern is that they are not deposited to GenBank. I can't access them from GISAID without permissions to their web pages. ****** 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: Leila Mousavizadeh Reviewer #2: No Reviewer #3: Yes:** Anastasia Vlasova Reviewer #4: 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.0241942.r001
Revision 1
18 May 2021 Author Response Medical Research Council Unit The Gambia at LSHTM P.O Box 273 Banjul, The Gambia Dear sir, I wish to thank you for your time and effort in reviewing our manuscript title “Origin of imported SARS-CoV-2 strains in The Gambia identified from whole genome sequences submitted in your Journal. The authors have thoroughly thought about all the major comments made on the manuscript and decided to change the paper to a short communication. Please find below answers (in red) to the concerns highlighted by the reviewers. 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 I have formatted the manuscript using the guidelines highlighted above. 2. We note that you are reporting an analysis of a microarray, next-generation sequencing, or deep sequencing data set. PLOS requires that authors comply with field-specific standards for preparation, recording, and deposition of data in repositories appropriate to their field. Please upload these data to a stable, public repository (such as ArrayExpress, Gene Expression Omnibus (GEO), DNA Data Bank of Japan (DDBJ), NCBI GenBank, NCBI Sequence Read Archive, or EMBL Nucleotide Sequence Database (ENA)). In your revised cover letter, please provide the relevant accession numbers that may be used to access these data. For a full list of recommended repositories, see http://journals.plos.org/plosone/s/data-availability#loc-omics or http://journals.plos.org/plosone/s/data-availability#loc-sequencing The genomes are now deposited at GenBank with the following submission number: SUB9545765 3. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (i) whether consent was informed and (ii) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information. If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information. Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”). For additional information about PLOS ONE ethical requirements for human subjects research, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research. This is sorted accordingly 4. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. [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: Partly Reviewer #3: Partly Reviewer #4: Yes 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: N/A Reviewer #3: No Reviewer #4: 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: No Reviewer #4: 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 Reviewer #3: Yes Reviewer #4: 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: Dear author(s), I have read your submission and it was interesting. Only there are minor things that you should consider for having better insight to your study. You may pay attention to this point that whether or not this study has been done previously by others and compare your results. Another point that you should consider is that what is your main goal for future of performing this work and what was your expectation out of that data. Reviewer #2: In this paper, Kanteh et al. reported whole genome sequence of SARS-CoV-2 strains detected in Gambia and their phylogenetic analysis with other strains outside of the country. Given little viral sequence information from African countries, the study provides us with a clue to elucidate the global transmission dynamics of the virus at the relatively early time point of the current outbreak. However, I have several concerns in the manuscript to be addressed before the publication. Major points 1. Absolute “origin” or “source” cannot be inferred from phylogenetic tree. That is because there must be other viral strains from other countries that are more closely related to Gambian strains but unsampled and not included in their phylogenetic analysis. The reviewer is very right about this point. However, at the time of analysis, we ran all the sequences against the available genomes on Nextclade (n=4645) and constructed a phylogenetic tree. Despite the number of genomes added, the origin or source still remains the same as seen in the image below. 2. Related to the point #1, viral sequences from other countries included in the phylogenetic analysis are very few. There should be many more available sequence data of viral strains collected by March 2020 from all over the world in GISAID, ViPR, and so on. Yes, this is true. We actually ran our samples against other genomes from Nexstrain, however, we still have similar topology. Selection of genomes from GISAID was based on our pateint’s travel history. 3. Difference in sequences between Illumina and Nanopore protocols should be done by not phylogenetic trees but direct comparison of SNPs (number and positions) between them. I agree with you. However, if there was huge difference in terms of the number of SNPs from each platform, this would perhaps alter the topology of the tree. However, this was not seen except in one sample, where the Illumina data had a longer branch length that the Nanopore. We found that there were more SNPs from the Illumina than the Nanopore data. 4. Related to the point #3, the authors had better show either of only Figure 2 or Figure Given that both trees look similar, we decided to include only one in the manuscript. We also added a phylogenetic tree showing all the genomes in Nextclade between march and April 2020. Minor points 5. There are a lot of typo in the manuscript. Please read the manuscript carefully and correct them before re-submission. This has been corrected. 6. (Lines 34 and 203) “Wuhan reference genome” is not a scientific term. Please indicate the actual strain name as described in the line 177. Okay. This is sorted. 7. Texts in the lines 76 and 77-78 are redundant. This is sorted accordingly. 8. The names of genes for the screening and confirmation should be specified here (as described in the Table 2.) 9. I personally think Figure 1 is unnecessary and can be removed from the manuscript. Yet, I will leave the decision to the editor and authors. 10. In the table 2, there is “Ct” in the third column title. However, I do not see any Ct values in the table. 11. (Line 249) WGS should be spelled out. Reviewer #3: The authors conducted complete genomic sequencing of the first 6 cases of SARS-CoV-2 in The Gambia to determine/confirm their origin/genetic characteristics using Illumina MiSeq and Nanopore platforms. The study addresses an important issue because the knowledge on the genetic characteristic of SARS-CoV-2 strains circulating in The Gambia/Adrica. The study also appears to be technically sound. The results presented/discussed are insufficient for a full-length research article. If no additional analysis (see below) is done, the submission must be converted to a short communication. Overall, the manuscript is too focused on technical details of both sequencing methods employed and presents very little detail on the genomic/molecular analysis of the Gambian SARS-CoV-2 strains. This has to be changed by mostly re-writing the manuscript. It is not clear why the two sequencing approaches had to be used: Illumina MiSeq and Nanopore. Although, the authors stated that both methods had their own advantages, it is not clear why the two methods were needed to just determine the strain origin. “While Illumina sequencing may be more accurate in determining within sample-diversity, Nanopore data can help with the understanding of the linkage between SNPs within individual virions”. The importance/relevance of determining within host/virion diversity in this study is not clear. It is well established fact that coronaviruses exist as quasispecies within the same host. The abstract needs to be re-written. Currently, it’s a mere statement of the methodology used in the study. It has to be re-written to properly convey the problem/background, approach, findings and conclusion of the study. Accession numbers for the newly generated sequences must be provided, but were missing. Issues with the analysis: More SARS-CoV-2 strains should be included in the phylogenetic trees. Phylogenetic distance and bootstrap values should be reflected on the trees. The meaning of the dashed lines should be explained in the figure legends. Recombination analysis should be conducted. Reviewer #4: This is a nice short paper on sequencing some SARS-CoV-2 genomes from Gambia. their methods seem fine, and the sequences seem reasonable. My only concern is that they are not deposited to GenBank. I can't access them from GISAID without permissions to their web pages. The genomes are now available at GenBank with the submission: SUB9545765 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: Leila Mousavizadeh Reviewer #2: No Reviewer #3: Yes: Anastasia Vlasova Reviewer #4: 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. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0241942.r002
28 May 2021 Decision Letter - Yury E Khudyakov, Editor PONE-D-20-33232R1 Origin of imported SARS-CoV-2 strains in The Gambia identified from whole genome sequences PLOS ONE Dear Dr. Kanteh, 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 revised manuscript was reviewed by 2 original reviewers. Both still identified serious problems in your revision. Please review the attched comments and provide carefully conceived responses. ============================== Please submit your revised manuscript by Jul 12 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: http://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, Yury E Khudyakov, PhD Academic Editor PLOS ONE [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 #2: (No Response) Reviewer #4: (No Response) ******** 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 #2: Partly Reviewer #4: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: N/A Reviewer #4: 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 #2: Yes Reviewer #4: No ****** 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 #2: Yes Reviewer #4: 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 #2: The revised manuscript has been improved to be concise and easy to follow. Still, I have three big concerns in the revised manuscript. Lines 95-99: Their finding that 7 SNPs that were observed by Illumina but not by Nanopore appeared only after 10 days of infection in the same individual seem surprisingly a big number to me. The authors argued that "The SNP winked by the Nanopore, might be due to higher accuracy on Illumina..." How could the authors exclude the possibility that there was something wrong in the Illumina run for the particular sample? Figure 1 and texts: Case F was totally missed in the tree and main texts. Figure 2 and texts: Although the authors included and indicated 3 Gambian sequences in the tree, they should include and show the sequences of all 6 cases. Besides, there is no explanation about the figure in the main texts at all. Reviewer #4: I still can't find the sequences.... "The genomes are now deposited at GenBank with the following submission number: SUB9545765" SUB9545765 is not an accession number. I went to the NCBI pages, and did a search against all of the databases, and could not find "SUB9545765" in any of their databases. The NCBI SARS-CoV-2 portal lives here: https://www.ncbi.nlm.nih.gov/sars-cov-2/ and there's about a half-million genomes in there, and here the accession number usually contains two letters, followed by six numbers - for example: FR988027 or OU032008 are both accession numbers. Another example, from a whole genome sequencing project looks like this: WGS of SARS-CoV-2 circulating in Spain GenBank Accession: ERX5596932 BioProject ID: PRJEB43166 SRA study link: ERP127101 SRA run number: ERR5956411 ****** 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 #2: No Reviewer #4: Yes:** David Ussery [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.0241942.r003
Revision 2
12 Jul 2021 Author Response This uploaded in Attach File section Attachments Attachment Submitted filename: response to Reviewers.docx https://doi.org/10.1371/journal.pone.0241942.r004
9 Aug 2021 Decision Letter - Yury E Khudyakov, Editor Origin of imported SARS-CoV-2 strains in The Gambia identified from whole genome sequences PONE-D-20-33232R2 Dear Dr. Kanteh, 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, Yury E Khudyakov, PhD Academic 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 #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 #2: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? 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 #2: (No Response) ****** 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 #2: (No Response) ****** 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 #2: (No Response) ****** 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 #2: No https://doi.org/10.1371/journal.pone.0241942.r005
Formally Accepted
16 Aug 2021 Acceptance Letter - Yury E Khudyakov, Editor PONE-D-20-33232R2 Origin of imported SARS-CoV-2 strains in The Gambia identified from whole genome sequences. Dear Dr. Kanteh: 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. Yury E Khudyakov Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0241942.r006

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