PONE-D-23-20026Efficient wastewater sample filtration improves the detection of SARS-CoV-2 variants: an extensive analysis based on sequencing parametersPLOS ONE

Dear Dr. Olivero,

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 authors are required to address the major and minor issues outlined by both Reviewer 1 and Reviewer 2. It is recommended that the authors consult a copyeditor to ensure that all typographical and grammatical have been addressed prior to resubmitting the manuscript. 
• The statistical analysis needs to be revised and clearly described in the methods section. The results of the statistical analysis needs to be stated under the results section.

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Academic Editor

PLOS ONE

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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: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: No

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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

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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: No

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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: The authors focused on the use of wastewater surveillance to not only monitor the level of infection within a population but also to use for the early detection of variants. This paper is a response to recommendations from the European Union Commission to establish an approach to establish WW surveillance of SARS-CoV-2 and its variants. The authors aimed to obtain high-quality sequencing data to allow variants detection and compared for sample conditions and evaluated the best condition by assessing several sequencing parameters. The authors provided an overview of sequencing parameters to consider when the final goal is to detect variants.

It is important to use WW to monitor the level of infection within a population, but WW is also needed for the early detection of variants as the clinical sequencing data will continue to focus on symptomatic patients. Surveillance, quantification, sequencing, and comparison of results is intricate as wastewater is a complex matrix. Therefore, this study is valuable and important. However, the statistical analysis of the results needs to be revised and explained further before the proposed conclusions can be validated. Additionally, the high viral load found in the samples studied make it difficult to extrapolate the proposed results to a more “normal” scenario. The following issues need to be addressed before publication:

Issues

1- Lines 240-245. To compare the read frequencies differences between the four experimental conditions for three biological samples, the authors use the student’s t-test. There are requirements for applying the t-test: 1- normal distribution of the data obtained however, the sample number is too low to predict normal distribution.2- is used for pairwise comparisons, however four conditions are being compared. ANOVA for multiple comparisons is more suitable. After using a proper statistic test the significance of the differences detected should be reassessed.

2- The authors should follow the MIQE guidelines for reporting their qPCR results. The standard curve, ranging from 102 copies (cp)/μL to 0.5 cp/μL that is generated by serial 10-fold dilution does not provide enough points.

3- The authors do not provide an explanation of why the DNAse treatment and concentration of RNA in filtered samples increases the % of reads corresponding to Bacteria.

Minor issues

1- Line 129 describes 2 μL of TNA or purified RNA from each extracted sample was used for the one-step RT-qPCR. However, line 136 mentions 5μL of TNA/RNA for each WW sample and standard point.

2- Line 170 mentions ANOVA Analysis of variance but the results mention t-test significance.

Reviewer #2: The authors have conducted a study comparing methods for preparation of wastewater samples for amplicon sequencing of SARS-CoV-2, in particular assessing how the quality of sequencing data is impacted by prefiltering wastewater and implementing a step for purifying and concentrating extracted RNA. The authors conclude that filtration of wastewater samples leads to improved specificity and coverage of SARS-CoV-2 sequencing data, while treatment of RNA samples reduced the percentage of sequencing reads aligned to SARS-CoV-2 but had little impact on other sequencing metrics.

Although the experimental design and metrics used to assess SARS-CoV-2 sequencing quality were valid, the statistical methods used in analysis require improvement and clarification to draw valid conclusions. In particular, the statistical tests need to take into account the fact that samples exposed to the different treatments were derived from the same three wastewater samples and should therefore be treated as matched samples. Authors should also carry out comprehensive statistical tests (e.g. two-way ANOVA) to compare all the treatments together before implementing pairwise comparisons as they have currently done. Furthermore, the manuscript contains multiple linguistics errors and typos which need addressing, possibly through the use of a copyeditor.

Considering this, I recommend that this manuscript is publishable with major revisions.

Major issues

- The statistical methods used in data analysis require revision and clarification. Currently, the authors have stated in the methods (line 169-170) that they used “Student's t-test and ANOVA Analysis of variance” for statistical analysis. However, there is no mention of the results of ANOVA in the manuscript associated with any of the figures, making it unclear whether these tests were carried out. Furthermore, as mentioned above, the replicates within each treatment were derived from the same three wastewater samples, meaning statistical tests should consider that these replicates are matched between treatments. Finally, the two treatment variables that the authors are trying to determine the effects of (filtration and RNA treatment) are applied consecutively to these samples and are therefore nested, meaning these shouldn’t be considered as four separate treatments of one independent variable. The impact of these variables should be assessed by two-way ANOVA (or non-parametric equivalent, depending if parametric assumptions are met) to determine the overall effect of the two variables and any interaction between them.

- The manuscript contains grammatical, linguistic and typographical errors, which require revision. I have detailed a few of these in the minor issues. I recommend that the authors could consult a copyeditor to resolve this.

Minor Issues

- Line 33 – it is stated that previous studies are “ignoring” wastewater filtering and RNA purification, despite these factors being addressed in multiple studies (as referenced). Consider revising this statement.

- Line 35 – more information should be given on the treatments i.e i) filtering wastewater and i) DNAse treatment, purification and concentration of RNA.

- Line 54 – please define “WW” again in the main text.

- Line 54 – revise grammar in “was proposed as an epidemiologic tool approach…”

- Line 59 – “transmission among asymptomatic has also” needs amending e.g. asymptomatic hosts.

- Line 78-79 – The statement “amplicon sequencing for viral WGS is a preferable approach to library preparation” requires revision, as amplicon sequencing involves library preparation. Please revise this to state which method of library preparation you are comparing amplicon sequencing to.

- Line 82-83 – Please revise the grammar in “but not many monitoring have been”

- Authors names need to be added where references are used as part of sentences in the text, or sentence structure amended accordingly. This occurs in multiple locations in the manuscript e.g. Line 88 “…studies conducted by 21-22 showed…”.

- Line 97 – amend typo “ae”.

- Line 116 – Please clarify whether the centrifugation step mentioned in this line is an additional step to the centrifugation described in line 107, or a repeated description of the same step? If there was only one centrifugation, please remove from line 116 for clarity.

- Line 142 – please give final concentration of RP primer or include stock concentration used for dilution.

- Line 145 – please remove repetition of “reaction”.

- Line 153 – please state the unit of concentration (ng/ul?).

- Line 165 – please clarify whether one million PE reads was the aim for sequencing depth or whether this normalisation was achieved during bioinformatic analysis.

- Line 177 – please detail which programs were used for trimming and consensus genome generation.

- Line 180 – Please clarify what “minimum frequency” refers to – possibly minimum frequency of variations?

- Lines 194-199 – section “as detailed…followed by purification and concentration” not required as this is already described in the methods section.

- Figure 1 and Table S2 – please indicate whether there were any significant differences between treatments in these metrics.

- Line 235-236 – please clarify what is meant by “in intra- and inter-conditions”.

- Figures 1/3/4/5/6 – These plots are referred to as histograms, when they are bar plots with categorical variables on the x-axis.

- Figure 1 – Consider replacing this with a plot representing the data in Table S2, as this is what makes up the majority of results discussed in this section.

- Figure 2 – The colours in this figure are not clear, probably due to the low resolution of the image which also makes the axis labels illegible.

- Line 259 – Please clarify what is referred to as “lower lines”.

- Line 263 – It is unclear what is meant by “genomic contamination” and how this would impair sequencing – please discuss further to clarify.

- Line 283 – it is unclear whether all samples had a percentage of reads classified as SARS-CoV-2 of less than 40%, or just the means for these treatments are <40%.

- Line 289 – Please explain further the speculation that RNA sample concentration would lead to increased bacterial reads.

- Line 296-297 – Could you provide some discussion as to why wastewater filtration may improve quality scores?

- Section 3.4.2.2 – The percentage of reads assigned to SARS-CoV-2 was much higher with CLC workbench than Kraken. It would be good to comment on why this is, possibly due to raw reads used in Kraken classification including many short reads which are filtered out before direct mapping.

- Figure S2 is redundant as the % reads not mapped can be extrapolated from % mapped reads.

- Line 343 – The difference in % mapped reads is only significant between F-NT and F-T, but not between NF-NT and NF-T

- Line 347 – amend “off-target reads” as these are not reads at this point e.g off-target nucleic acid fragments.

- Line 346-347 – This finding is contrary to Mondal et al 2021 who found RNA purification and DNase treatment to increase % reads aligned to SARS-CoV-2, and Child et al 2023 (https://doi.org/10.1371/journal.pone.0284211) who found RNA purification and concentration to also increase alignment rate. Please discuss your findings in relation to these studies.

- Line 375 – amend “essential” as low frequency variants are still identified in unfiltered samples.

- Line 388-9 – Could you provide statistical tests to back up the statement that “mutation frequency was higher in the F conditions”? Also, please discuss why the mutation frequency would be higher in filtered samples if these mutations are associated with the BA.1 variant, which is stated to be dominant in the samples. Could the unfiltered treatments be picking up on more diversity of variants than filtered treatments, reducing the frequency of these typical BA.1 mutations in favour of other variants?

- Line 402 – Please amend this statement, as only the F-NT treatment passed for all samples, not F-T.

- Figure 8 – The colour scale used in this figure requires clarification. These percentages are clearly not directly related to the percentage parameters in the “Sequencing parameters” column. Is this scale relative to the highest and lowest value for each parameter amongst the treatments?

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Reviewer #1: No

Reviewer #2: Yes: Harry T. Child

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PONE-D-23-20026R1Efficient wastewater sample filtration improves the detection of SARS-CoV-2 variants: an extensive analysis based on sequencing parametersPLOS ONE

Dear Dr. Olivero,

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 complete the minor revisions as given below, before the manuscript is fully accepted for publication. 

Please submit your revised manuscript by May 18 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Poovendhree Reddy

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Please ensure that the quality of your figures is improved in the next revision. 

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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)

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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: Yes

********** 

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: 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

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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

********** 

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 authors have conducted a study comparing methods for preparation of wastewater samples for amplicon sequencing of SARS-CoV-2, in particular assessing how the quality of sequencing data is impacted by prefiltering wastewater and implementing a step for purifying and concentrating extracted RNA. The authors conclude that filtration of wastewater samples leads to improved specificity and coverage of SARS-CoV-2 sequencing data, while treatment of RNA samples reduced the percentage of sequencing reads aligned to SARS-CoV-2 but had little impact on other sequencing metrics.

The experimental design and metrics used to assess SARS-CoV-2 sequencing quality are valid, and following amendments in response to the initial round of reviewers comments the statistical methods used are now valid and clear. Considering this, I recommend that this manuscript requires only minor revisions. I understand that these are largely grammatical suggestions, but I hope the authors appreciate the improvement in clarity that they can provide.

Minor Issues

- Figure legends – Two way ANOVA results are currently givenm in the format “Two-way ANOVA (* = p ≤ 0.05).”. Please amend these without the asterisk as otherwise it seems to be referring to the asterisks in the plot which are the paired T test results e.g. “Two-way ANOVA (p ≤ 0.05)”.

- Figure 2 – The resolution of Figure 2 is still very low in the version I have been sent. This should be checked before publication.

- Line 54 – Suggested revision of “Wastewater Based Epidemiology (WBE) was proposed as an epidemiological tool approach for wastewater (WW) surveillance” to “Wastewater Based Epidemiology (WBE) was proposed as an epidemiological tool approach for disease surveillance”

- Line 59 – “asymptomatic host” needs amending to “asymptomatic hosts”

- Line 80-83 – Suggested revision of “However, most of the viral genome sequences used to monitor viruses’ evolution have been“ to “However, most of the viral genome sequences used to monitor the evolution of viruses have been“, and “but not many monitoring have been performed” to “but not much monitoring has been performed”

- Line 87-89 – please amend to “Earlier than the development of WBE for the COVID-19 pandemic, previous studies showed that the detection of viral pathogens in sewage depended on the concentration and extraction methods used (21–23).”

- Line 248-249 – may I suggest an amendment to “Moreover, the number of viral cp input into library preparation was not identical within or between conditions...”

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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: Yes: Dr Harry T. Child

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[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.

Efficient wastewater sample filtration improves the detection of SARS-CoV-2 variants: an extensive analysis based on sequencing parameters

PONE-D-23-20026R2

Dear Dr. Olivero,

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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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,

Ricardo Santos

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

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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: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: 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

**********

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

**********

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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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: Yes: Harry T. Child

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