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PONE-D-20-40848

Deep sequencing of DNA from urine of kidney allograft recipients to estimate the donor-specific DNA fraction

PLOS ONE

Dear Dr. Belkadi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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he authors need to address all reviewer's comments:

Reviewer # 1:

The authors have a creative concept that the degree of kidney allograft rejection can be estimated by deep sequencing DNA found in recipient urine.  An analysis of the SNV frequencies is then proposed as a means of predicting the relative contributions of donor versus recipient DNA; the hypothesis is that, with increasing recipient DNA contribution, one can infer an increased proportion of responding immune cells and therefore a higher likelihood of allograft rejection.

The concept seems promising, but I fear that the authors gloss over some significant caveats to this method. The first is the assumption that recipient-specific DNA originates mostly from tissue-invading immune cells; as the authors note, they were able to previously perform such an analysis on kidney recipients with urinary tract infections and showed lower proportions of donor DNA when individuals were diagnosed with UTI (compared to individuals without). This does seem to indicate that recipient-specific DNA can come from recipient immune cells responding to a urinary tract infection. In a clinical setting, do you anticipate being able to tell the difference between a low donor-specific fraction (as a result of low rejection) and a low donor-specific fraction (as a result of competition by infiltrating immune cells due to infection)?  This would be particularly relevant for subclinical UTIs.

Secondly, I would caution the authors against stating that they can determine the donor-specific DNA fraction, given that this method is unable to determine which DNA specimen is from which individual. I agree with their interpretation that the fraction of recipient to donor cells SHOULD be higher for AR than for ATI (and for no-pathology specimens), however, as they state, that future complementary study would be necessary.

Specific questions about the procedure:

1)     Could the authors address why they chose to use the Breast cancer risk panel rather than other available genomic sequencing panels?

2)     Does this method use cell free DNA, or does it include a cell lysis step? Are the authors concerned about the relative proportions of donor/recipient DNA in the cell free vs. cellular fractions?

3)     Regarding DNA target enrichment due to skewed amplification, I am curious about the SNVs that are NOT in the primer regions. I presume that, if one of the amplification primers fails to bind effectively due to a SNV, it affects the results not just of that SNV but of all SNVs within that amplicon? I understand and wholeheartedly agree with the authors’ decision to exclude SNVs falling within primer sequence regions; I wonder if the quantification of other SNVs is negatively affected by these binding issues as well.

I appreciate the commitment to patient privacy, and do not expect the authors to share specific point mutations for their study subjects.  However, the manuscript would benefit from the inclusion of some additional data, for example, concentrations of extracted DNA both before and after library preparation, specific numbers for the donor-specific fraction of each tested patient, etc.

Overall, the authors should be commended on a well-written paper that clearly expresses their concepts and logical process. Some sentences are awkwardly written and could benefit from a second look, but it doesn’t reach the level of incomprehension

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PLOS ONE

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Reviewer #1: Partly

Reviewer #2: Yes

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

Reviewer #1: I Don't Know

Reviewer #2: I Don't Know

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: The authors have a creative concept that the degree of kidney allograft rejection can be estimated by deep sequencing DNA found in recipient urine. An analysis of the SNV frequencies is then proposed as a means of predicting the relative contributions of donor versus recipient DNA; the hypothesis is that, with increasing recipient DNA contribution, one can infer an increased proportion of responding immune cells and therefore a higher likelihood of allograft rejection.

The concept seems promising, but I fear that the authors gloss over some significant caveats to this method. The first is the assumption that recipient-specific DNA originates mostly from tissue-invading immune cells; as the authors note, they were able to previously perform such an analysis on kidney recipients with urinary tract infections and showed lower proportions of donor DNA when individuals were diagnosed with UTI (compared to individuals without). This does seem to indicate that recipient-specific DNA can come from recipient immune cells responding to a urinary tract infection. In a clinical setting, do you anticipate being able to tell the difference between a low donor-specific fraction (as a result of low rejection) and a low donor-specific fraction (as a result of competition by infiltrating immune cells due to infection)? This would be particularly relevant for subclinical UTIs.

Secondly, I would caution the authors against stating that they can determine the donor-specific DNA fraction, given that this method is unable to determine which DNA specimen is from which individual. I agree with their interpretation that the fraction of recipient to donor cells SHOULD be higher for AR than for ATI (and for no-pathology specimens), however, as they state, that future complementary study would be necessary.

Specific questions about the procedure:

1) Could the authors address why they chose to use the Breast cancer risk panel rather than other available genomic sequencing panels?

2) Does this method use cell free DNA, or does it include a cell lysis step? Are the authors concerned about the relative proportions of donor/recipient DNA in the cell free vs. cellular fractions?

3) Regarding DNA target enrichment due to skewed amplification, I am curious about the SNVs that are NOT in the primer regions. I presume that, if one of the amplification primers fails to bind effectively due to a SNV, it affects the results not just of that SNV but of all SNVs within that amplicon? I understand and wholeheartedly agree with the authors’ decision to exclude SNVs falling within primer sequence regions; I wonder if the quantification of other SNVs is negatively affected by these binding issues as well.

I appreciate the commitment to patient privacy, and do not expect the authors to share specific point mutations for their study subjects. However, the manuscript would benefit from the inclusion of some additional data, for example, concentrations of extracted DNA both before and after library preparation, specific numbers for the donor-specific fraction of each tested patient, etc.

Overall, the authors should be commended on a well-written paper that clearly expresses their concepts and logical process. Some sentences are awkwardly written and could benefit from a second look, but it doesn’t reach the level of incomprehension.

Reviewer #2: I'm really sorry,but I must say that I'm not competent to review this paper despite the the fact that researchfield is my field (kidney) and the paper seems very interesting (that's why I accepted to be reviewer), but when I read the methods and results parts, there is too much informatic and mathematic for me.

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Reviewer #1: No

Reviewer #2: No

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Deep sequencing of DNA from urine of kidney allograft recipients to estimate  donor/recipient-specific DNA fractions

PONE-D-20-40848R1

Dear Dr. Belkadi,

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

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

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Kind regards,

Stanislaw Stepkowski

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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