Whole genome sequence analysis showing unique SARS-CoV-2 lineages of B.1.524 and AU.2 in Malaysia

Ummu Afeera Zainulabid; Aini Syahida Mat Yassim; Mushtaq Hussain; Ayesha Aslam; Sharmeen Nellisa Soffian; Mohamad Shafiq Mohd Ibrahim; Norhidayah Kamarudin; Mohd Nazli Kamarulzaman; How Soon Hin; Hajar Fauzan Ahmad

doi:10.1371/journal.pone.0263678

Peer Review History

Original SubmissionAugust 10, 2021
15 Oct 2021 Decision Letter - Ahmed S. Abdel-Moneim, Editor PONE-D-21-25859Whole Genome Sequencing Analysis of Spike D614G Mutation Reveals Unique SARS-CoV-2 Lineages of B.1524 and AU.2 in MalaysiaPLOS ONE Dear Dr. AHMAD, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Nov 29 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. 3. Please upload a new copy of Figure 2 as the detail is not clear. Please follow the link for more information: " ext-link-type="uri" xlink:type="simple">https://blogs.plos.org/plos/2019/06/looking-good-tips-for-creating-your-plos-figures-graphics/" " ext-link-type="uri" xlink:type="simple">https://blogs.plos.org/plos/2019/06/looking-good-tips-for-creating-your-plos-figures-graphics/" 4. 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. Is the manuscript technically sound, and do the data support the conclusions? Review Comments to the Author Reviewer #1: General comment Considering the large scarcity of sequences from this country (but not limited to), the work of Zainulabid et al, about SARS-CoV-2 molecular epidemiology in Malaysia, comes to contribute with CoV-2 genomic surveillance studies. The work is logically well conducted and written. Considering the SARS-CoV-2 spread flow for other countries vs the intense individuals movement across the globe, the work also adds novelty by inferring and discussing the new lineages circulating. Although this Referee feels the lack of time-scale trees to try establish the origin of these new strains, on the other hand, considering the current pandemic scenario, the work brings interesting and useful data for the scientific community. Minor concerns Materials and Methods Sample selection Please, inform us the protocol used for detection of SARS-CoV-2 RNAs extraction Line 123: Please, add the ct value. Public database SARS-CoV-2 genome analysis Line 149: Does the criteria of sequence selection have been based only on the length? What about the sequencing coverage? Please, add such information. Results - Could you please clarify the results from Table 3? It seems paradoxical. - Please, improve the quality of all figures presented. - Fig. 2. Phylogenetic trees provided are difficult to read. - Fig. 2 C and D - please, provide the whole tree. - Fig. 3: What is A and B? Where is the start and end position? It is confusing. The way it is, it does not add too much to the work. - Fig. 4. same above (Fig. 3) - Fig. 5. Did you mean Spike TM Domain? Origin of the massive spread of COVID-19 cases in Pahang - Sometimes it is difficult to understand what the authors are inferring because of the codes such as IIUM5763/2021. Maybe replacing it by the city(district)/year (code between parenthesis) would become easier to the readers. The same for the trees (tip label). - Please, replace and add the WHO new nomenclatures for SARS-CoV-2 variants since it becomes easier to understand --- in the whole manuscript. Discussion Line 350: Please, use “our analysis of” instead of “our detailed analysis of”. Line 358: add a reference Line 358: same from line 350. Line 436: Please, choose to use countries or cities. Reviewer #2: The paper is well written and results are novel. However, some grammatical errors needs the attention e.g. line 63 had (has), line 203 were (was), line 249 is (was), line 250 is (was), line 306 was (is), line 340 was (are), line 383 follows (followed) etc. Another concern is that the sample size is small. Please justify it. Add mechanism underlying the mode of action of SARS-CoV-2 in causing the disease and symptoms. ********** [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.0263678.r001
Revision 1
14 Dec 2021 Author Response 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 Response: Thank you for providing the template. Our revised manuscript follows the journal guideline to the best of our understanding. 2. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. Response: The data has been submitted to the GISAID public domain and accessible to all who wish to access. Accession numbers of sequences are also provided in the manuscript and in the phylogenetic analysis and figures. 3. Please upload a new copy of Figure 2 as the detail is not clear. Please follow the link for more information: https://blogs.plos.org/plos/2019/06/looking-good-tips-for-creating-your-plos-figures-graphics/" https://blogs.plos.org/plos/2019/06/looking-good-tips-for-creating-your-plos-figures-graphics/" Response: Figure 2 has been thoroughly revised for clarity and now represent more detailed and resolved information. 4. 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. Response: We have thoroughly scanned the list of references and no such reference (retracted) was found. We have also transformed the references as per PLoS One format. Citations of all papers which were then in preprint and now published are accordingly modified. Reviewers' comments: Comments to the Author Reviewer #1: General comment Considering the large scarcity of sequences from this country (but not limited to), the work of Zainulabid et al, about SARS-CoV-2 molecular epidemiology in Malaysia, comes to contribute with CoV-2 genomic surveillance studies. The work is logically well conducted and written. Considering the SARS-CoV-2 spread flow for other countries vs the intense individuals movement across the globe, the work also adds novelty by inferring and discussing the new lineages circulating. Although this Referee feels the lack of time-scale trees to try establish the origin of these new strains, on the other hand, considering the current pandemic scenario, the work brings interesting and useful data for the scientific community. Response: We are grateful for the reviewer suggestion and in the phylogenetic analysis presented in the revised manuscript we have address this observation. Minor concerns Materials and Methods Sample selection Comment: Please, inform us the protocol used for detection of SARS-CoV-2 Response: SARS-CoV-2 was detected through RT PCR and mentioned under Sample selection heading RNAs extraction Comment: Line 123: Please, add the ct value. Response: Samples with less than 25 ct values were selected for the subsequent investigation and this information has also been provided in the revised manuscript. Public database SARS-CoV-2 genome analysis Comment: Line 149: Does the criteria of sequence selection have been based only on the length? What about the sequencing coverage? Please, add such information. Response: Criteria of sequence selection has been elaborated and highlighted in the same section in the revised manuscript. “Sequences were selected based on completion of the genome with minimum number of unresolved nucleotides. Restraining the selection from the total of 1356 to 986 sequences” Results Comment: Could you please clarify the results from Table 3? It seems paradoxical. Response: Table 3 shows the prediction of three different programs to suggest the potential impact of the mutations. Since all three programs based on different algorithms and used different base line data, it is possible, rather not very uncommon that they may end up giving different predictions for same mutation. Comment: Please, improve the quality of all figures presented. Fig. 2. Phylogenetic trees provided are difficult to read. Fig. 2 C and D - please, provide the whole tree. Response: Figure 2 has been thoroughly revised for clarity and now represent more detailed and resolved information. Phylogenetic tree with more pertinent sequences was reconstructed with Wuhan as an outgroup with relatively more statistical support of 1000 bootstraps (revised version) compared to 500 (previous version). Whole tree is also provided in MEGA format for the reference as supporting information. Comment: Fig. 3: What is A and B? Where is the start and end position? It is confusing. The way it is, it does not add too much to the work. Response: We are highly grateful and agree to the suggestion of the reviewer, since the information is already present in the text of the manuscript, we have removed the figure 3. Comment: Fig. 4. same above (Fig. 3) Response: We are highly indebted for the suggestion; Figure 4 has been modified for better clarity and respective legend of the figure elaborate the flow of the Figure 4 (previous version) and now Figure 3 (revised version). In the schematic diagram of spike protein N and C terminal are also labelled. Comment: Fig. 5. Did you mean Spike TM Domain? Response: Yes, full name is also used in the figure legend Origin of the massive spread of COVID-19 cases in Pahang Comment: Sometimes it is difficult to understand what the authors are inferring because of the codes such as IIUM5763/2021. Maybe replacing it by the city(district)/year (code between parenthesis) would become easier to the readers. The same for the trees (tip label). Response: Indeed, this is an excellent suggestion and we have made two important changes in this regard. 1. In the revised manuscript and in the phylogenetic analysis figure (Fig 2) we have used GISAID accession number instead of sample code along with the geographical location of Pahang SARS-CoV-2 D614G variants. 2. In the revised manuscript we have also added the collection month and year of the Pahang SARS-CoV-2 D614G variants Comment: Please, replace and add the WHO new nomenclatures for SARS-CoV-2 variants since it becomes easier to understand --- in the whole manuscript. Response: In the revised manuscript WHO nomenclature is used where appropriate Discussion Comment: Line 350: Please, use “our analysis of” instead of “our detailed analysis of”. Response: Modifications made Comment: Line 358: add a reference Response: Reference added Comment: Line 358: same from line 350. Response: Modifications made Comment: Line 436: Please, choose to use countries or cities. Response: We are grateful to the reviewer for the suggestion and modifications are made in the revised manuscript Reviewer #2: The paper is well written and results are novel. However, some grammatical errors needs the attention e.g. line 63 had (has), line 203 were (was), line 249 is (was), line 250 is (was), line 306 was (is), line 340 was (are), line 383 follows (followed) etc. Response: We are thankful to the reviewer for the suggestion and modifications are made in the revised manuscript Comment: Another concern is that the sample size is small. Please justify it. Response: Sample selection criteria has been explained in the methodology section of the revised manuscript. Comment: Add mechanism underlying the mode of action of SARS-CoV-2 in causing the disease and symptoms. Response: We are thankful to the reviewer for highlighting this, brief description regarding the SARS-CoV-2 mode of action has been added in the introduction. Attachments Attachment Submitted filename: Reponse to Reviewers.docx https://doi.org/10.1371/journal.pone.0263678.r002
25 Jan 2022 Decision Letter - Ahmed S. Abdel-Moneim, Editor Whole Genome Sequence Analysis Showing Unique SARS-CoV-2 Lineages of B.1.524 and AU.2 in Malaysia PONE-D-21-25859R1 Dear Dr. AHMAD, 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, Ahmed S. Abdel-Moneim, Ph.D. Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0263678.r003
Formally Accepted
18 Feb 2022 Acceptance Letter - Ahmed S. Abdel-Moneim, Editor PONE-D-21-25859R1 Whole Genome Sequence Analysis Showing Unique SARS-CoV-2 Lineages of B.1.524 and AU.2 in Malaysia Dear Dr. Ahmad: 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. Ahmed S. Abdel-Moneim Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0263678.r004

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