PONE-D-21-09048

Non-invasive prenatal paternity testing by analysis of Y-chromosome mini-STR haplotype using next-generation sequencing

PLOS ONE

Dear Dr. Ming Liu,

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.

I would suggest that you read this following articles which will be of help to answer the reviewers' comments: 

1) Wang et al. STR polymorphisms of "forensic loci" in the northern Han Chinese population. Journal of Human Genetics July 2003, Volume 48, Issue 7, 337-341

2)  Tamaki et al. Microsatellite typing in a paternity case against a deceased man whose two brothers were available for testing.  Jpn J Leg Med 1996 50(2) 82-86

Please submit your revised manuscript by October 11. 2021. 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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We look forward to receiving your revised manuscript.

Kind regards,

Wei Wang, M.D., Ph.D.

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: Partly

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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: No

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript describes a strategy for non-invasive prenatal paternity testing by analyzing the Y-chromosome. The method is useful and should be published following revisions (both major and minor) as indicated below (pages and line refer to the PDF format of the manuscript):

MAJOR COMMENTS

Materials and methods, under “DNA extraction”, page 4

1. For the following statement:

“The quantity and purity of the extracted DNA were determined by sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with Tanon 1600 Gel Imaging System (Shanghai Tanon Science & Technology, Shanghai, China) and NanoDrop 2000 spectrophotometry (Thermo Fisher Scientific, Waltham, MA, USA) respectively”:

provide reference or describe in more detail the methods used. In addition, provide information as to how much of the 60 μl extract was used for SDS-PAGE, for quantitation and for subsequent NGS library preparations.

2. Did the authors use any negative and/or reagent blank controls during DNA extractions and subsequent operations?

Materials and methods, under “Library preparation and NGS”, page 5

3. “DNA extracted from cell-free maternal plasma…”: Provide information as to how many μl of the cell-free DNA extract were used.

4. “…was amplified at the following 12 Y-chromosome mini-STR loci”: Provide the sequence information on the mini-STR primers that were designed for the present assay. In addition, usually barcoding in NGS is used for sample designation, therefore, it is essential that more details should be provided as to the NGS assay with respect to both the samples and the Y-STR loci assayed. More details are required to describe the developed NGS assay.

5. “The quantity and purity of the DNA libraries were assessed by SDS-PAGE.”: Provide reference for the method or more details.

Materials and methods, under “Calculation of paternity testing parameters”, page 6

6. “…the frequency of the son’s haplotype, µ is the mutation rate, n is the total number of Y-STR haplotypes,…”: Provide in this paragraph (i.e. in Materials and methods) the information regarding the use of a mutation rate of 0.00123 for locus DYS393 (indicated on page 8 of the MS under the Results section).

7. Also, another paragraph should be added to indicate that the CPIs were calculated based on 6 loci Y-haplotypes. In other words, the information provided at the beginning of page 10 of the manuscript: “Given the limited information on the Y-STR loci haplotypes in the Liaoning population, only 6 out of the 12 loci tested in our study could be used for calculating CPI and probability of paternity” should be provided in the Materials and Methods section.

Results, pages 6-8

8. The Results section must include (either in Table format or descriptive) information on the quantities of the 24 cell-free fetal DNA extracts as determined by the methods indicated in the Materials and Methods section.

9. As indicated before, a small note, preferably in the Materials and methods section, should indicate that the haplotype frequencies in Table 1 (page 8 of the manuscript) were estimated using the haplotypes for 6 Y-chromosomal loci.

Discussion

10. The inclusion of an internal control to verify that the lack of amplification is due to the absence of the Y-chromosome and not to experimental failure is an important aspect especially in real cases. Perhaps the Amelogenin locus, that has small amplicons could also be included in the assay. Authors to elaborate briefly on this (i.e. how to distinguish failure Vs absence of Y-chromosome) in the discussion section.

References

11. Please go over the references and make sure that authors, journals etc are cited correctly. For example Reference 14 should be corrected to read: Roewer L, Andersen MM, Ballantyne J, Butler JM, Caliebe M, Corach D, ME, Gusmão L, Hou Y, de Knijff P, Parson W, Prinz M, Schneider PM, Taylor D, Vennemann M, Willuweit S (2020) DNA commission of the International Society of Forensic Genetics (ISFG): Recommendations on the interpretation of Y-STR results in forensic analysis. Forensic Science International: Genetics 48: 102308

MINOR COMMENTS/SUGGESTIONS

Abstract

12. Suggestion to revise the sentence, page 1, to read: “The Y-chromosome mini-STR genotypes of all 14 male cffDNA were obtained…”

13. Suggestion to revise the sentence, page 2, to read: “The combined paternity index (CPI) and probability of paternity calculation was based on 6 loci Y-haplotype distributions of a local population.”

Introduction, pages 2

14. Suggestion to revise the sentence, page 2 (last s lines), to read: “Due to its uniparental inheritance, the results of Y-STR typing are interpreted on the basis of haplotype…”

Introduction, page 3

15. Suggestion to revise the sentence, page 3 (1st line), to read: “A major challenge in non-invasive prenatal paternity testing (NIPPT) is the detection of fetal-specific markers of the paternal genetic material…”

Materials and methods, page 4

16. Suggestion to revise the sentence, page 4 (under "DNA extraction", 1st line), to read: “A total of 5 mL peripheral blood was collected from the pregnant women in anticoagulant-treated tubes.”

17. Suggestion to revise the title to read: “Paternity testing by Capillary Electrophoresis (CE)”

Results, page 8

18. Suggestion to revise the title of the Table to read: “Table 1. The paternity testing parameters using cffDNA in the 14 cases with male fetuses”

Discussion, page 9

19. Suggestion to revise the sentence, page 9 (1st sentence - since the beginning of sentence spell out CE) to read: “Capillary Electrophoresis is the routine technique…”

20. Suggestion to revise the sentence, page 9 (lines 2-3), to read: “…genetic information that it can provide and process, high throughput…”

21. Suggestion to revise the sentence, page 9 (line 13), to read: “…millions of SNPs have to be analyzed due to low PIC (polymorphism information content) leading to statistics with lower power. In addition…”

22. Suggestion to revise the sentence, page 9 (lines 14-15), to read: “…SNPs in forensics is controversial since some SNPs may be present in the coding regions with bioethical implications stemming from confidentiality and privacy concerns since they may disclose sensitive information or are modified due to disease [29].”

23. Suggestion to revise the sentence, page 9 (lines 16-17), to read: “Therefore, given the limitations of CE and SNP-based NGS, mini-STR-based NGS with high testing capacity and high PIC is a better choice for NIPPT.”

24. Suggestion to revise the sentence, page 9 (last 2 lines), to read: “Due to strong linkage disequilibrium, the multiplying of single locus allele frequencies cannot be used for Y-STR CPI calculation.”

Discussion, page 10

25. It is recommended to add a small paragraph in Materials and Methods to describe the population that was used to calculate the haplotype frequencies in relation to what is written in the Discussion (page 10; lines 3-4): “In 7 of the 14 cases, the fetal haplotype were not detected in the 838 Liaoning haplotypes reported by Guo et al [22]. According to the ISFG algorithm [14]…”:

26. Suggestion to revise the sentence, page 10 (lines 19-21) to read: “...Y-chromosome mini-STR should be used for unwanted pregnancy as the result of sexual assault or for sex selection in case of gene defect and inherited disease.”

References, page 11

27. Suggestion to change the title to: “References” instead of “Reference”

Reviewer #2: Song et al. propose a proof-of-concept study in which they use massively parallel DNA sequencing to assign paternity to male fetuses in utero. The authors use CPI analysis and compare the alleged father's Y-chromosome STR haplotype to the fetus' haplotype, which is obtained noninvasively from the mother's blood plasma. The manuscript is well-written and the main ideas are clear.

Major comments:

While this study can be considered a valid contribution to science, my main concern is related to how the authors have interpreted their results. I’m not convinced that using only the Y-STR haplotype is sufficient to assign paternity with confidence for two reasons.

First, even though a CPI is calculated to show some degree of uncertainty to the paternity tests performed, we should keep in mind that the Y-STR haplotype represents only one locus in the genome. Thus, this Y-STR paternity analysis would be similar to accepting the use of only one autosomal locus as sufficient to determine paternity with confidence (which can also show greater than 99% CPIs depending on the locus, frequencies and alleles scored).

Second, unlike the autosomal loci, the Y-chromosome is inherited from father to son without recombination. This mode of inheritance creates the potential for several men in the population to share the exact same Y-STR profile (i.e. they belong to the same lineage). For instance, if the paternal grandfather of one of those fetuses had four brothers and each of those brothers (including the grandfather himself) had four sons, and each of those 16 men had two sons, we would have, in this family alone, 52 individuals that would, in theory, match the fetus’ Y-STR profile. This isn’t the case when analyzing autosomal STR profiles (with at least a dozen loci each) due to recombination. In other words, only identical twins are expected to share the same STR profile in the population while several men can (and do) share the same Y-STR haplotype, creating the real potential of false positive results.

For these reasons, unlike it’s implied in the title and throughout manuscript, Y-STR haplotype results alone should not be used to confirm paternity. Instead, it could be used to “exclude” or “not exclude” a male from being a potential father of a fetus. Especially in the forensic setting, where paternity tests can have broad impacts on people's lives, conclusions should always be confirmed with more reliable methods such as genotyping multiple autosomal STR loci.

Minor comments:

1) In the Abstract, where it’s said “The cffDNA genotype was validated by the paternal genotype” I believe the word “genotype” should be replaced by “haplotype” as this sentence seems to be related to the Y-chromosome. I also suggest this wording is checked throughout the manuscript.

2) At the end of the Abstract and the Discussion, the authors mention “sex selection”. This is a very controversial subject due to its ethical implications. And as the authors mention, such practice is forbidden in many countries. Should this potential application be promoted or even discussed in this publication?

3) Materials and Methods (Sample collection): I suggest rewording the first sentence this way: “Peripheral blood samples were collected from 24 pregnant women undergoing prenatal tests, and their male partners, at the Dalian Blood Centre from April 2018 to December 2019”. Dalian Blood *Centre* or Dalian Blood *Center*?

4) Materials and Methods (DNA extraction/library preparation and NGS): Assessing the quantity and purity of DNA extracts and library preparations with SDS-PAGE doesn’t seem to be standard practice. Can you describe a little more how this is done?

5) Figure 2 (flowchart): the boxes “Infant STR genotypes” and “Paternal STR genotypes” appear to be swapped.

Reviewer #3: The study applied a 12 Y-STR multiplex to pre-natal testing of foetal DNA in maternal circulation. Standard statistical paternity testing regimes were applied to confirm paternity. A novel aspect of the work is the use of NGS sequencing to genotype the STRs. However, no primer details or sequence output analysis is given - the Results section is extremely thin, as if the authors assume that the readership would not be interested in how well the NGS assay for these 12 Y-STRs worked with such low-level DNA input. The authors do not discuss their choice of Y loci - why select these 12?

No description is given for the extent to which sequence variation could be used and potentially was of value in the paternity analyses made. This was because normal CE was used to type the matched father's DNA, and NGS for the maternal samples, whereas, it would be potentially better to establish how much sequence variation could benefit the paternity statistical analyses in each case. Therefore, the authors lost every opportunity to report in this paper the sensitivity of the system they have developed in terms of sequence coverage and expanded identification discrimination from sequence variants in the chosen loci.

As such, the work was made scientifically, but there is little of novelty or impact in the study (and I realise the paper cannot be rejected on these grounds alone).

Figures 1 and 2 are largely redundant.

An initial text slip suggested an English review would be beneficial, but in fact, the standard of the rest of the paper is fine:

Our proof-a-concept study demonstrated that Y-chromosome mini-STR can be used > Our proof-of-concept study demonstrated that Y-chromosome mini-STRs can be used.

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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.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Christopher Phillips

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PONE-D-21-09048R1Non-invasive prenatal paternity testing by analysis of Y-chromosome mini-STR haplotype using next-generation sequencingPLOS ONE

Dear Dr. Liu,

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 Dec 30 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: 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,

Kelvin Yuen-Kwong CHAN, Ph.D.

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.

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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 #1: All comments have been addressed

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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 #1: (No Response)

Reviewer #2: Dear colleagues:

In this current version, I believe that Song et al. have addressed most of the criticisms raised by the reviewers in the first submission. However, I'm still VERY CONCERNED about the fact that the authors are proposing a paternity test based exclusivelly on Y-STR haplotypes. In fact, my major comments, which I copy again below, were not responded by the authors.

I would like to stress that unlike autosomal STR genotypes, IDENTICAL Y-STR haplotypes are found in the population because the Y-chromosome is passed on from father to son without recombination. Thus, I strongly suggest that the authors at least acknowledge in the manuscript the possibility of false-positive results due to the inheriteance mode of Y-STR haplotypes. And ideally, the authors could also recommend that the Y-STR results must be confirmed with autosomal STR markers when a sample can be safely collected from the fetus/baby (e.g. after birth or miscarriage).

FROM PREVIOUS REVIEW:

First, even though a CPI is calculated to show some degree of uncertainty to the paternity tests performed, we should keep in mind that the Y-STR haplotype represents only one locus in the genome. Thus, this Y-STR paternity analysis would be similar to accepting the use of only one autosomal locus as sufficient to determine paternity with confidence (which can also show greater than 99% CPIs depending on the locus, frequencies and alleles scored).

Second, unlike the autosomal loci, the Y-chromosome is inherited from father to son without recombination. This mode of inheritance creates the potential for several men in the population to share the exact same Y-STR profile (i.e. they belong to the same lineage). For instance, if the paternal grandfather of one of those fetuses had four brothers and each of those brothers (including the grandfather himself) had four sons, and each of those 16 men had two sons, we would have, in this family alone, 52 individuals that would, in theory, match the fetus’ Y-STR profile. This isn’t the case when analyzing autosomal STR profiles (with at least a dozen loci each) due to recombination. In other words, only identical twins are expected to share the same STR profile in the population while several men can (and do) share the same Y-STR haplotype, creating the real potential of false positive results.

For these reasons, unlike it’s implied in the title and throughout manuscript, Y-STR haplotype results alone should not be used to confirm paternity. Instead, it could be used to “exclude” or “not exclude” a male from being a potential father of a fetus. Especially in the forensic setting, where paternity tests can have broad impacts on people's lives, conclusions should always be confirmed with more reliable methods such as genotyping multiple autosomal STR loci.

Reviewer #3: All required clarifications and explanations in the text have been made. The description of the figures as 'redundant' was meant to signify that they served no purpose and could be excluded - but their retention is not a problem. I understand the underlying NGS data will be made available in due course, even thought the authors cannot meet this obligation at the moment.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Christopher Phillips

[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.

Non-invasive prenatal paternity testing by analysis of Y-chromosome mini-STR haplotype using next-generation sequencing

PONE-D-21-09048R2

Dear Dr. Liu,

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.

Please kindly take note the following comments from Reviewer #2 when revising your manuscript:

1) Abstract - This sentence my be missing a period: "The cffDNA haplotype was validated by the paternal haplotype The paternity testing parameters were attributed to each case quantitatively."

2) Figure 2 (and throughout the manuscript) - In some cases it's unclear when the authors are talking about Y-STR HAPLOTYPES or AUTOSOMAL STR GENOTYPES since they did both (e.g. Fig. 2). To better distinguish those, I suggest that when talking about Y-STRs the word "haplotype" is used and when talking about autosomal STRs, the word "genotype" or the phrase "autosomal genotype" is used.

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,

Kelvin Yuen-Kwong CHAN, Ph.D.

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: 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: Abstract - This sentence my be missing a period: "The cffDNA haplotype was validated by the paternal haplotype The paternity testing parameters were attributed to each case quantitatively."

Figure 2 (and throughout the manuscript) - In some cases it's unclear when the authors are talking about Y-STR HAPLOTYPES or AUTOSOMAL STR GENOTYPES since they did both (e.g. Fig. 2). To better distinguish those, I suggest that when talking about Y-STRs the word "haplotype" is used and when talking about autosomal STRs, the word "genotype" or the phrase "autosomal genotype" is used.

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Reviewer #2: No