PONE-D-20-14518

Use of amplicon-based sequencing for testing fetal identity and monogenic traits with single circulating trophoblast (SCT) prenatal diagnosis

PLOS ONE

Dear Dr. Zhuo,

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.

All reviewers expressed major concerns regarding the manuscript. It is required that you address all these concerns so that we may further consider this manuscript.

Please submit your revised manuscript by Sep 20 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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

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

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Osman El-Maarri, Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If the need for consent was waived by the ethics committee, please include this information.

3. We note that Figure(s) [1] in your submission contain copyrighted images. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a.    You may seek permission from the original copyright holder of Figure(s) [1] to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b.    If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

4. Thank you for including the following competing interests statement; "The Houston authors are faculty and staff at Baylor College of Medicine (BCM), which is a partial owner of a for profit diagnostic company, Baylor Genetics (BG); Houston authors also are employee of or have advisory or lab director roles at BG. ALB is founder and CEO of Luna Genetics, Inc."

Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests).  If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

[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: Partly

Reviewer #2: Partly

Reviewer #3: No

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: I Don't Know

**********

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: Dear editor,

The paper “Use of amplicon-based sequencing for testing fetal identity and monogenic traits with

single circulating trophoblast (SCT) prenatal diagnosis” by Zhuo et al. is well written and clearly describes a promising procedure to distinguish fetal from maternal cells. However, I have a few remarks that require clarification.

Major point

- There is no discussion on the presence of fetal cells still present from previous pregnancies. Fetal cells have been detected decades after a pregnancy. Can the method described also distinguish if a fetal cell has a different haplotype than other fetal cells? If so, this is a strength of the methods described. If not, this would be a limitation.

- In addition, what would be the effect of twin pregnancies?

- Extra discussion is needed on the effects of the analysis on downstream analyses. Does the procedure cause lower quality results in downstream analysis of selected cells, since extra WGA amplification is needed to perform this test

Minor points,

- Line 999: Settings used for BWA-MEM. Does ‘conventional BWA-MEM’ mean all default settings?

- Table 1 is rather a list of amplicons containing SNP regions than a list of SNPs.

- In table 1: the start or stop of the HLA-A amplicon seems to be incorrects (Start>stop and not matching amplicon size).

- Lines 321 and 322, perhaps add the Protein accession numbers to the variants

- One method to infer haplotypes in cfDNA was not yet mentioned. https://pubmed.ncbi.nlm.nih.gov/28844486/

- Spelling mistake in fig 4: mother � mother and (informatic � informative?)

- The github code would benefit from some polishing, adding comments and removing commented out codes.

Reviewer #2: In this paper, the authors study the application of WGA followed by SNP and / or haplotype analysis in multiple amplicons to determine the fetal or maternal origin of presumably fetal cells, retrieved from the maternal blood. They do so by using different amplicons, i.e. amplicons from regions such as the HLA regions, with multiple SNPs per amplicon for haplotyping, and Human Identification SNP test amplicons, with one SNP per amplicon. They also studied the possibility of using these methods for genotyping for monogenic traits. Below my comments and questions:

1. My main comment is that, even though the authors do mention the drawbacks of their method, the paper is quite optimistic. The theory behind the methods they describe is solid, but the actual data are still quite preliminary. In their abstract the authors say that the method allowed reliable differentiation between fetal and maternal cells, in the introduction in line 75 they mention “in most cases”, but this was only in cases with sufficient information, which is certainly not 100%. Not all data is presented in a clear way, such as numbers of cells that are informative, but in how many cases? See my comments below. The methods that are described certainly have potential for this application, but need to be improved and the authors do acknowledge this in the discussion. In the discussion, the authors do mention multiple options for improvements, but how feasible are these? If feasible, why didn’t the authors already implement these, or at least show some data of improved versions, as proof-of-principle. The authors show data on fetal genotyping for monogenic traits, but in the discussion they mention themselves that a low failure rate is to be expected due to failure to isolate fetal cells and/or to ADO.

2. Throughout the paper, the authors use the term cell-based NIPT, whereas in the title they use the term single circulating trophoblast prenatal diagnosis. I would suggest to use the same terminology throughout the paper, and would suggest to also use the term cell-based NIPT in the title.

3. Line 40: analysis cell free = analysis of cell-free

4. In the introduction, the authors mention some drawbacks of cf-NIPT. One major drawback, of course, is the fact that cf-NIPT studies cd-DNA from the placenta and not from the fetus itself. This major drawback is not solved by the cell-based technique the authors use, as trophoblast cells are used for this method. The authors should comment on this. Furthermore, even though it is outside the scope of this paper, in the introduction the authors should also (briefly) comment on drawbacks of the cell-based NIPT, such as costs as compared to cf-NIPT and high-throughput possibilities.

5. With SNP typing, DNA from 156 blood samples were compared, with data from 518 cells: 357 cells were informative. Does being informative mean that the cells were fetal, or were there also maternal cells identified? If all fetal, were these cells originally also from 68.9% of the 156 samples, or were cells from some samples overrepresented and others underrepresented? In other words, could informative (fetal?) cells be retrieved from 68.9% of the 156 samples? (This refers to the comment that not all data support the conclusions, questions 1 and 3 of the review).

6. For haplotyping, how many cells from how many samples were tested? Again 156 samples? Also here, were the informative cells distributed evenly over the samples. In how many samples could fetal cells be identified based on haplotyping only? Is 50% of all cells the same as 50% of the cases? (Again, see questions 1 and 3 of the review)

7. The authors state they tested the haplotyping method with matched gDNA, WBC and fetal cells. In Line 278, they only mention WBC, not the fetal cells. What is the differentiating rate for the fetal cells? Please mention this in the text, not only in a supplementary figure, as this is the main subject of this paper. Moreover, according to Figure S3A, this is about 50% for both WBC and fetal cells, and this percentage is determined by one amplicon (HLA-B).

8. Figure S3A shows fetal cells with a detection rate of about 50%. In Figure S3B for HAP this seems to be more than 60%. What is the difference?

9. Lines 302-304: the authors state that 5 cells were genotyped: two were heterozygous and two had ADO. What happened to the fifth one?

10. In line 288, the authors state that in the cultured lymphoblasts, the ADO rate was 15% and 8%. When using true fetal cells from the three families, the ADO is very much higher, about 50%, as in each family some cells show ADO. How do the authors explain this difference?

11. From two of the three families more cells were retrieved than were used for genotyping. Why did the authors not genotype all available cells? Could not all cells be identified as true fetal? If so, that the identification of true fetal cells is lower than 60-70%. How were these cells identified as true fetal? If the other cells were maternal in origin, the chance of picking up a maternal cell is much higher than 10%, as stated in the introduction in line 65.

Reviewer #3: This paper describes the use of SNPs genotyping by sequencing in order to identifiy circulating cells of fetal origin in the context of NIPT applications.

Although the subject matter and the approach are interesting, there are significant issues with the way some of the results are presented and especially with the conclusions reached by the authors.

Because of the major limitations of the proposed methods, highlighted by several results (e.g. the combined performance of SNPs genotyping and haplotyping that gives only a 70% rate of success for identifying fetal cells from WBC (line 278, fig S3 B), a high rate of allele dropout and amplification artefacts contributing to false-negatives and false positives (line 335-341)), a clear and detailed presentation of false positive/negative rates should be provided.

Unfortunately, these rates are not comprehensively and clearly presented in the paper. Table 2 is not clearly explained. For example, I'm puzzled by the apparently negative correlation between true positives (TPV) and % of SNP differences (diff SNP%) in the "Fetal cell" column, shouldn't it be the opposite ? More info about the table headers, interpretation, etc. should be given. One of the main focus of the paper should be to present, analyse and discuss these rates in details.

This relatively poor performance of the genotyping approach to confirm fetal origin seems to be caused at least in part by the low amount/quality of starting DNA material since the detection rate for genomic DNA is much higher (Fig S3 A). This impact of starting material is also evident from the much lower number of scorable SNPs from WGA products as compared to genomic DNA shown on fig. 2.

These results thus point to serious technical challenges associated with WGA from single cells. Moreover, as the author acknowledge at line 335-341, both a high rate of allele dropout and amplification artefacts contribute to false-negatives and false positives.

The allele dropout problem mentionned above was also seriously affecting the genotyping for monogenic diseases, with rates reaching 8-15% (line 288). Such rate is clearly incompatible with any diagnostic application.

From these results, the realistic take home message is clearly one of skepticism towards the feasibility of using this kind of approach clinically in the short to mid term. However, although the authors acknowledge some of these limitations in the discussion, they nevertheless make statements that I consider misleading with respect to what the data show. For instance, "This single cell genotyping assay can provide an essential step for confirming the fetal origin of cells obtained from cell-based NIPT workflows. Through genotyping, we can reject cells that are of indeterminate or maternal origin and provide metrics for WGA DNA quality using multiple amplicons." (line 328-331). "...this assay is rapid and reliable..." (line 335). "Use of this method for genotyping fetuses at risk for specific monogenic mutations is feasible." (line 352-353).

More details should also be provided about the exact number of amplicons used rather than vague statements such as: “We sequenced approximately 90 highly polymorphic SNPs within about 40 amplicons.” (line 72). Likewise, in the methods section, three groups of amplicons are mentioned but only for the second group, a number is provided (37), while a reference is offered for the first group (Debeljak et al.), no number is given concerning the third group. Table 1 listing the amplicon used contains 49 entries.

Overall, the way the methods and results are presented lacks clarity and details that complicates the evaluation of the technique.

I thus consider that in its current form, this paper suffers from 1) a lack of clarity and a lack of emphasis on false positive/negative rates and other performance statistics, and 2) needs a more sober and realistic interpretation of the results, less focused on optimistic hopes of better future performances and more concerned about current challenges.

**********

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: Lennart F. Johansson

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-20-14518R1

Use of amplicon-based sequencing for testing fetal identity and monogenic traits with single circulating trophoblast (SCT) as one form of cell-based NIPT

PLOS ONE

Dear Dr. Zhuo,

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 Feb 18 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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

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

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Osman El-Maarri, Ph.D

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

Reviewer #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 #2: Partly

Reviewer #3: Partly

**********

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

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 #2: Yes

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 #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 #2: The authors have done a significant amount of work from which the paper certainly benefits.

Previously, I commented on the difference between cells and cases. Even though the data in the paper are presented in a much better way now, I still have some questions:

1. In the rebuttal the authors mention the number of cases is now 154 instead of 156. In the text, they use the number of 154, while in table 2 the numbers do not add up to 154 but to 152. Please explain or correct.

2. In Table 2, numbers of cases are presented, which in my opinion is indeed the most informative way of (at least) presenting the data. In lines 213-218 the authors again switch to information on number of cells instead of cases. This apparently is not the same as cases, as when calculating % from the data in Table 2, different % are obtained than mentioned in the text. Please use the same way of presenting data in both text and Table.

3. In Table 2 the last column seems to be the same as the “2 SNP + 6%” column as far as data is concerned. What does this last column show? According to Table 3 it should be “2 SNP + 0%”, but what does that mean? The numbers in the column in Table 2 are the same as in the “2 SNP + 6%” column. Is this correct?

4. Table 2 would benefit from mentioning also the percentages, for better comparison with the text.

5. Lines 226-230 and Table 3: this is again based on cells. Please also provide information of cases.

6. Lines 337-342: As mentioned in my previous comment, the data n the numbers of cells do not add up to the numbers of cells that are genotyped. Please add information on all the cells, for both the DHCR7 and the sickle cell family.

7. Line 337: “…but the data are somewhat limited…”. Please remove the word “somewhat”, as this single word raises a lot of questions and removing the word does not change the message of the sentence.

8. Lines 403-405: the authors suggest as alternatives to overcome the problem with their method to use their NIPT in combination with amniocenteses and CVS. This seems a strange alternative, as cell-based NIPT is being validated and optimized as replacement for invasive methods. What would be the benefit of cell-based NIPT if it should be used in combination with an invasive procedure anyway?

Minor comments:

9. Abstract line 26: “This method allowed reliable differentiation of fetal and maternal cells.” This is still an optimistic, and possibily misleading, sentence. The sentence can be removed without any damage to the abstract.

10. Throughout the paper, the term “mutation” is used. There is an international agreement to use the word “variant” or “pathogenic variant” instead (see Richards et al. 2015). Please use this throughout the paper.

11. Line 199: “…where none the cells…” = “…where none of the cells…”

12. Line 202: “…meaning that no…” = “…meaning no….”

13. Line 214: “…used a more conservative….” = “…used a more conservative approach of…”

14. Line 226: “…two different 2 SNP…” = “two different SNPs…”

15. Line 241: “…per sample…” = “…per cell…”

16. Line 351: “…the sickle mutation…” = “…the sickle cell anemia variant…”

17. Line 377: spelling error in pregnancies and piology (=etiology??)

Reviewer #3: Although the authors have attempted to address all the comments raised in the previous review, a few issues persist.

Notably, in my opinion, the discussion still contains overly optimistic statements that do not adequately portrait what the current data suggest. For instance, at line 381, I would replace: "...this assay is rapid and reliable…" by "...this assay is promising…".

at line 393, replace: "...monogenic mutations is feasible." by "...monogenic mutations is potentially feasible.". At line 410, replace: "An improved version of this assay may significantly reduce…" by "A future improved version may hopefully reduce…".

The paper would also greatly benefit from clarifying the terminology used when referring to "informative" cells and SNPs throughout. It seems like the authors refer to SNPs with more than one allele present in the NGS data from a sample as an informative SNP, whether or not there's a difference between the mother and the fetus at this SNP position (e.g. when both mother and fetus are heterozygotes for the same alleles), while an informative cell refers to a cell with feto-maternal SNP differences. These terms should be formally defined early on to avoid confusion. However, at line 299, the authors refer to informative SNPs as those where there's a difference between the genotype of the mother and of the putative fetal cell. The term scorable SNP is also used, which here seems to simply refer to a genotyped SNP position that passed quality control.

Additional minor suggestions and comments

line 98: "The first step is conventional PCR with a pool of amplicons with bridging adaptors…" should be : "The first step is conventional PCR of a pool of amplicons with bridging adaptors…"

line 140: "An 8-10 pM diluted denatured…"

line 188: "...three SNPs show a difference between one of the cell…"

line 198: I believe it should be three groups and not four: "The results for cases can be divided into three groups…"

line 214: "...we initially used a more conservative criterion of at least…"

line 215: "We found that 68.9 % of genotyped putative fetal cells…"

line 228: what does precision of 96.1 % refers to here ? To the authors mean global diagnostic accuracy ? Please mathematically define the precision calculation used.

line 235: "The remaining three cells…"

line 348: The last sentence ("The fetus does carry the paternal mutation.") seems odd and should be removed or modified.

**********

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

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

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

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-20-14518R2

Use of amplicon-based sequencing for testing fetal identity and monogenic traits with single circulating trophoblast (SCT) as one form of cell-based NIPT

PLOS ONE

Dear Dr. Zhuo,

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 Apr 23 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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

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

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Osman El-Maarri, 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.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: (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: Yes

Reviewer #3: Yes

**********

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

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 #2: Yes

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 #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 #2: (No Response)

Reviewer #3: The authors have addressed most of the issues previously identified, but there remains a few details that need to be clarified.

As requested, the authors have added a sentence defining and clarifying the "informative/uninformative" terminology in the methods section, where this definition is given: "Throughout this manuscript, a cell or a SNP is referred to as informative if the putative fetal cell has an allele not carried by the mother...".

However, at line 249, while refering to Fig. 4, they state that "Cell G106 has only 2 SNP difference in less than 20 informative SNPs...". The axis on Fig. 4 are indeed labelled "number of SNPs not present in mother" and "number of informative SNPs", for the y and x axis respectively.

Therefore, given the definition of informative SNP provided by the authors, I don't understand what can be the difference here between "number of SNPs not present in mother" and "number of informative SNPs". Aren't SNPs not present in mother suppose to be alleles not present in mother ? Please clarify.

Regarding Fig. 4, there are other inconsistencies between the text and the figure. At line 245, it is said that cells G78, G212, G227 and G232 all have at least four SNPs wich are different from maternal gDNA while on the graph, it seems to be at least five SNPs which are different (of these four cells, G78 and G212 have the lowest number of differences and it seems to be five differences when judging from the axis and the points on the graph). Then it is said that the remaining three cells had 0-2 SNPs which differed from maternal gDNA, while it seems to be rather 0-1 judging from the figure ?

Moreover, the caption for Fig. 4 says: "As expected, the maternal gDNA samples gave no alleles not present in the mother...". This sentence seems odd given that thoughout the paper, we get the impression that maternal gDNA (from WBC) is used as the reference for the mother's genome. How could there be an allele not present in the mother from the data used to establish the mother's genome ? That would be circular ? Was there another reference used ?

Finally, at line 100, the requested change of "...conventional PCR with a pool..." for "...conventional PCR of a pool..." has not been made, even if the authors claim to have made the change in their reply.

**********

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

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

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

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Use of amplicon-based sequencing for testing fetal identity and monogenic traits with single circulating trophoblast (SCT) as one form of cell-based NIPT

PONE-D-20-14518R3

Dear Dr. Zhuo,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Osman El-Maarri, Ph.D

Academic Editor

PLOS ONE