PONE-D-22-35643Multidimensional Scaling Methods Can Reconstruct Genomic DNA Loops Using Hi-C Data PropertiesPLOS ONE

Dear Dr. Ishibashi,

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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: Yes

Reviewer #2: Partly

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: 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: No

Reviewer #2: Yes

Reviewer #3: No

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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

Reviewer #3: 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 author demonstrated an application of the multidimensional scaling (MDS) method to Hi-C data for visualizing DNA loops. In general, Hi-C data have information on population-averaged 3D chromatin structure, and the data format finally becomes a matrix of the contact frequency. First, the author transformed the contact matrix into a distance matrix. Then, the author tried to extract a 3D genome structural feature using the MDS method relating to the singular value decomposition. Next, he/she defined criteria to find DNA loops. Interestingly, his/her proposed method revealed that DNA loops form at active and open chromatin regions, which is consistent with a current viewpoint on the loop domain. Therefore, the reviewer found that the scientific treatments sound good, and these quantitative results would be reliable. On the other hand, the reviewer would like to require revisions based on the following comment:

1. In the main text, the author uses "ChIP-seq" and shows a table of the analysis of H3K27ac, which is related to the transcriptionally active state. However, the author did not mention the chromatin state or motivation to analyze ChIP-seq data. The author should mention such information.

2. How did the author find the logarithmic weight in Eq. (1)? What is the base of the logarithm? If the base is 5, the equations should make sense.

3. The base of the logarithm of the Y-axis in Figs. 1 and 2 should be described.

4. The reviewer could not understand the data format in Supplementary Files. For example, the file "GSE141067(osteosarcoma)/0min/SVD_234/plot.chr1.0-4980.txt" has 5 columns and 2989 rows.

5. l. 130 and Eq. (3): The author converted the contact frequency into the distance without any references. Although the assumption is not theoretically supported in terms of polymer physics [Shinkai et al. NARGAB 2020], some computational methods have conventionally assumed [Serra et al. FEBS Lett 2015].

6. Fig. 3 legend must be incomplete. The author only plotted the eigenvectors. Moreover, the figure includes blue lines without explanation.

7. What does the author mean by "roots" in Fig. 4 legend?

8. Eq. (7): The variable "V" seems to be a new one. What is the difference between "v" and "V"?

9. l. 174-183: Can this algorithm uniquely determine loop regions? This definition must be the most critical part of this work. However, the reviewer could not understand it and Fig. 4 correctly. A schematic of the algorithm would help broad readers.

10. p. 6: Please remove the Fig.5 legend.

11. l. 207: The author describes, "The number of DNA loops and number of genes are close to each other," referring to Table 1. However, the number of DNA loops is almost 30000, and the number of genes is almost 5000 in Table 1. These are NOT close to each other.

12. §3.2: Has the author analyzed H3K27me3 ChIP-seq data? The histone marker is related to heterochromatin.

13. Figure 7 is not attached to the manuscript.

Reviewer #2: In this manuscript, Ryo utilizes multidimensional scaling (MDS) to capture DNA loops for high-throughput chromosome conformation capture (HiC) data. By transforming HiC data to a distance matrix, MDS identified DNA loops contained more DNA bound by transcription factors than existing methods. The manuscript is well organized.

I have 4 major concerns.

1. In section 2.2.2, the term “exaggerated representations of DNA loops” is confusing. Could you clarify the definition and rationale behind it? I am also concerned about the threshold of 50kbp for formula 1. Would the significant loops of length 50kbp be affected by this transformation?

2. In section 2.2.3, the authors claimed that the second and the third eigenvectors often retain the original structure. Empirically, how would one decide for real HiC data which eigenvectors to use for identifying loops? Why is the first eigenvector excluded?

3. In formula 8, the distance matrix is smoothed with a window size of 100kbp. Has the author evaluated the performance of MDS varying different window sizes? For other HiC data, how would the user recommend selecting this hyperparameter?

4. In table 1, is 318380 the number of all potential DNA loops considered? What do the ratios in two columns represent? # of loops seems much bigger than # of genes in table 1, which contradicts the statement in lines 207-210, “the number of loops and number of genes are close to each other?” Could you quantify this?

I have 3 minor comments.

1. It would be helpful to have a table of datasets to summarize the read depth, bin size, year, and HiC data type.

2. Below line 195, the description of figure 5 should be revised.

3. The grammar and wording can be much improved for this manuscript.

Reviewer #3: Ishibashi suggested a method (MDS) to predict DNA loops based on Hi-C sequencing data. It tested on 7 publicly available Hi-C datasets from various cancers (e.g., breast cancer, lymphoma, and kidney cancer). However, there are four major problems in the manuscript:

1) In the Methods section, the description of MDS in predicting DNA loops is not so clear. The algorithm, code and demo data for this study shall be publicly available for others, in order to reproduce the results and understand the method. The prediction accuracy for DNA-loops has to be rigorously evaluated by proper statistical methods.

2) In the Results section, the presentation of predicted loops in each dataset is too short to evaluate its merit. In Particular, the results have to be compared with other similar tools for predicting DNA loops by using Hi-C sequencing datasets. And also, I am not clear about how other genomic information (e.g., enhancer or promoter histone markers and nucleosome density) at these predicted loops, which are important markers for validating the predicted DNA loops/interactions, are functional or random interactions.

3) In the Discussions section, the author needs to improve this part significantly to convince people that the proposed new method is useful for studying DNA loops based on Hi-C data.

4) Finally, the quality of all main figures is not good, which has to be improved significantly.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PONE-D-22-35643R1Multidimensional Scaling Methods Can Reconstruct Genomic DNA Loops Using Hi-C Data PropertiesPLOS ONE

Dear Dr. Ishibashi,

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.

There are minor issues to be further addressed. please submit the revised manuscript

Please submit your revised manuscript by Jun 12 2023 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,

Divijendra Natha Reddy Sirigiri

Academic Editor

PLOS ONE

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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:

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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

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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 #1: Yes

**********

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

Reviewer #1: 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

**********

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: No

**********

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: The author revised all concerns of the reviewer. However, there are still points that need to be clarified according to the following comments:

Major points:

1. Equations (4) -- (6).

Although the author describes a general framework for the eigenvalue decomposition or the singular value decomposition, it is still unclear how to convert the matrix D into the vector X (= (x_1, x_2, ..., x_N) ?). Furthermore, the definition of the second and the third eigenvectors are insufficient. The order of the corresponding eigenvalues is not defined.

2. p6. l175.

The author should understand that E_i does NOT have the physical unit because the matrix V is a unitary matrix, and the eigenvectors are normalized without the physical unit. Therefore, E_i is NOT the "physical" distance.

3. p7. ll191--196.

The definition and algorithm of the root were improved and readable. More simply put, is the location of the local minimum of E^-_i the root?

Typos:

1. p2. l23.

stoHi-C -> The stoHi-C

2. p4. l105.

Fig 1 shows -> Figure 1 shows

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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.

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Reviewer #1: No

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Multidimensional Scaling Methods Can Reconstruct Genomic DNA Loops Using Hi-C Data Properties

PONE-D-22-35643R2

Dear Dr. Ishibashi,

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.

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Kind regards,

Divijendra Natha Reddy Sirigiri

Academic Editor

PLOS ONE

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