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

The SARS-CoV-2 disease, first detected in Wuhan, China, in December 2019 has become a global pandemic and is causing an unprecedented burden on health care systems and the economy globally. While the travel history of index cases may suggest the origin of infection, phylogenetic analysis of isolated strains from these cases and contacts will increase the understanding and link between local transmission and other global populations. The objective of this analysis was to provide genomic data on the first six cases of SARS-CoV-2 in The Gambia and to determine the source of infection. This ultimately provide baseline data for subsequent local transmission and contribute genomic diversity information towards local and global data. Our analysis has shown that the SARS-CoV-2 virus identified in The Gambia are of European and Asian origin and sequenced data matched patients' travel history. In addition, we were able to show that two COVID-19 positive cases travelling in the same flight had different strains of SARS-CoV-2. Although whole genome sequencing (WGS) data is still limited in sub-Saharan Africa, this approach has proven to be a highly sensitive, specific and confirmatory tool for SARS-CoV-2 detection.

Introduction

The emergence and re-emergence of pathogens such as severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) pose a grave threat to human health [1] The SARS-CoV-2 disease, first detected in Wuhan, China, in December 2019 has become a global pandemic [2] and is causing an unprecedented burden on health care systems and the economy globally [3-6]. The estimated number of cases has increased exponentially [6], especially in Europe and America, with significant but variable case-fatality rates inter-continentally. By April 27th, 2021, there were over 148 million SARS-CoV-2 confirm cases and 3.12 million deaths globally [7]. The Gambia, a tourism hotspot, reported more than five thousand SARS-Cov-2 cases and 173 deaths [7].

While the travel history of index cases may suggest the origin of infection, phylogenetic analysis of isolated strains from these cases and contacts will increase the understanding and link between local transmission and imported strains. The phylogenetic analyses of global SARS-CoV-2 sequences provide insight into the relatedness of strains from different areas and suggest the transmission of four super-clades, [8] geographically clustering into viral isolates from Asia (China), US (two super clades) and Europe. SARS-CoV-2 pose significant risk to global health, hence the need for effective strategies to detect the sources of infections, outbreaks and transmission patterns in different geographical settings. The objective of this analysis was to provide genomic data on the first six cases of SARS-CoV-2 in The Gambia and to determine the source of the strains. This would ultimately provide baseline data for monitoring subsequent local transmission(s) and contribute genomic diversity information towards local and global data.

Materials and methods

The Scientific Coordinating Committee at Medical Research Council Unit The Gambia at LSHTM has approved the manuscript to be publish given that Ethics Committee approval is not required to publish routine public health data.

Oxford Nanopore (GridION) and Illumina (MiSeq) platforms were used to sequence the viral genomes as summarised in Table 1. Total RNA was purified from Nasopharyngeal-Oropharyngeal (NP-OP) swabs using QiaAmp viral RNA mini kit (Qiagen– 52906). Library preparation was done following the ARTIC sequencing protocol [9] with little modification for Illumina sequencing. FASTQ files were analysed following the ARTIC bioinformatics pipeline [9] to generate consensus sequences. Phylogenetic tree was constructed using IQTREE (v1.3.11.1) [10] and visualised and annotated using Interactive Tree of Life (ITOL) (v5) [11]. Selection of genomes from GISAID for comparison with the isolated strains was based on patients’ travel history and the major geographical spread of the pandemic.

Table 1

Sample information for COVID-19 sequenced cases from The Gambia.

Case ID	Travelled from	Date Reported	Current Status	Number of samples submitted	Time points	Library prep type			Sequencing
						Depletion	ARTIC amplicon (NEB)	ARTIC amplicon (ONT -LSK109)	Illumina (MiSeq)	Nanopore (GridION)
A	London	16/03/20	Recovered	4	Days 0,4,7,10	2	4	4	4	4
B	Bangladesh	19/03/20	Dead	1	Day 0	0	1	1	1	1
C	France	20/03/20	Recovered	1	Day 0,	0	1	1	1	1
D	France	26/03/20	Active	2	Day 0,11	0	1	2	1	2
E	Netherland	23/03/20	Active	2	Day 0,14	0	1	2	1	2
F	Italy	13/03/20	Recovered	1	Day 0	0	1	1	1	1
Total				11		2	9	11	9	11

Cases A-D = Confirmed RT-PCR COVID-19 cases.

Case E = Indeterminate by RT-PCR.

Case F = RT-PCR COVID-19 negative.

Cases A and D travelled to The Gambia in the same flight.

Cases C and D both travelled from France.

Note: Status of patients given here was at the time of sequencing.

Results and discussion

Whole genome sequencing data was generated from the first six confirmed cases in the Gambia to determine the source of these strains as well as provide baseline data for subsequent local transmission. We also sought to assess if Nanopore sequencing platform, a portable and cheap sequencer can produce high quality genomic data with accurate SNPs for phylogenetic inference when compared to Illumina.

Our analysis showed that six genomes (4 samples from Case A, 1 from case C and 1 from case D) from the Gambian samples clustered with the European (Spanish and United Kingdom) SARS-CoV-2 strains Fig 1. This correlates with the patients’ travel history as they had been in Europe before arriving in The Gambia. Strains from cases C and D, both of whom travelled from France, were more closely related to the Spanish strain. Interestingly, cases D and A travelled to The Gambia on the same flight, however, their strains clustered on different nodes, indicating that they could have been infected independently, before the start of their journeys. Case F was a negative SARS-CoV-2 sample thus excluded from the phylogenetic analysis. Although only few genomes were selected elsewhere for our phylogenetic inference as shown in Fig 1, similar topology was observed when genomes deposited on Nextstrain [12] a public repository for SARS-CoV-2 sequences were included as shown in Fig 2.

Fig 1

A maximum likelihood phylogenetic tree of Gambia SARS-CoV-2 genomes and 11 SARS-CoV-2 strains isolated in different parts of the world.

Fig 2

A maximum likelihood phylogenetic tree of Gambia SARS-CoV-2 genomes isolated from The Gambia and those found elsewhere.

The tree was constructed using Nextclade.

Although viruses are known to mutate and change rapidly [13], the viral genome of case A collected at different time-points clustered on the same node indicating the patient had been shedding the same virus with no observed polymorphism according to our Nanopore data. The same sample sequenced on the MiSeq resulted in a longer branch length at day10 when compared to other time points. SNP analysis showed seven more SNPs on day10 sample compared to the same sample sequenced on the MinION (Nanopore data). This was an interesting finding, however, the SNP winked by the Nanopore, might be due to higher accuracy on Illumina or perhaps might as well be a sequencing error. This was difficult to ascertain give that the sample was run only once using both platforms.

The viral genome from case B who initiated travel from Bangladesh and then across four other countries, including Senegal, before arriving in The Gambia, clustered with a strain from Japan. This case may have contracted the virus in Asia and his travel history suggests he could have contributed to infections in other countries. The two isolates from case E at different time points clustered with strains from Japan as well. Interestingly, case E samples were indeterminate by rRT-PCR diagnostics. The indeterminate diagnostic rRT-PCR result could be due to low sensitivity of the assay, an indication of low viral density of SARS-CoV-2 in the sample. Therefore, subsequent follow up for such indeterminate cases is imperative for accurate information and aid our understanding of the disease progression as well as the evolution of this novel virus strain under different case management environments.

Summary

Our analysis has shown that the SARS-CoV-2 strains identified in The Gambia are of European and Asian origin and sequence data matched patients’ travel history. In addition, we were able to show that two COVID-19 positive cases travelling in the same flight had different strains of SARS-CoV-2. Although whole genome sequence (WGS) data of SARS-Cov-2 in sub-Saharan Africa is still limited, this approach has proven to be a highly sensitive, specific and confirmatory tool for SARS-CoV-2 detection. Hence, the use of second and third generation sequencing technologies coupled with bioinformatics will convincingly provide data for monitoring transmission dynamics.

We have also demonstrated that the Nanopore platform with its flexibility for number of samples per run, and the generation of data in real-time and at a reasonable cost makes it most suitable for outbreak monitoring, especially in resource limited areas. This would go a long way in providing knowledge on the molecular epidemiology of this disease, give the true burden of the disease in resource limited setting as well as provide information for African specific mutations which could have a significant implication on vaccine development and roll out.

6 Jan 2021

PONE-D-20-33232

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

PLOS ONE

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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

**********

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Reviewer #1: Yes

Reviewer #2: N/A

Reviewer #3: No

Reviewer #4: N/A

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: No

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Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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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.

**********

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Reviewer #1: Yes: Leila Mousavizadeh

Reviewer #2: No

Reviewer #3: Yes: Anastasia Vlasova

Reviewer #4: No

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18 May 2021

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:

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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

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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.]

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Submitted filename: Response to Reviewers.docx

Click here for additional data file.

28 May 2021

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.

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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.

==============================

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Yury E Khudyakov, PhD

Academic Editor

PLOS ONE

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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

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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.

12 Jul 2021

This uploaded in Attach File section

Submitted filename: response to Reviewers.docx

Click here for additional data file.

9 Aug 2021

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.

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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)

**********

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Reviewer #2: No

16 Aug 2021

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