PCOMPBIOL-D-26-00605

Deciphering chromatin architecture and dynamics in Plasmodium falciparum using the nucDetective pipeline

PLOS Computational Biology

Dear Dr. Laengst,

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Kind regards,

Vladimir B Teif, Ph.D.

Academic Editor

PLOS Computational Biology

Shaun Mahony

Section Editor

PLOS Computational Biology

Additional Editor Comments (if provided):

Thank you very much for the opportunity to read in detail your manuscript and responses to previous peer-reviews from Review Commons. I have invited the peer-reviewers who previously reviewed your manuscript at Review Commons to respond. Reviewer 1 has provided a detailed peer-review, but reviewers 2 and 3 were not available. Therefore, I have done my own reading of the current version of the manuscript and evaluated your responses to reviewers 2 and 3. Based on my reading, as well as the response of reviewer 1, which is included separately, this manuscript requires a minor revision, as detailed below.

1) Please address the minor revision suggestions from Reviewer 1.

2) Reviewer 2 asked several relevant questions in relation to the NRL change. I can see your substantial response to this reviewer’s point in the response letter, including a fragment size histogram plot that the reviewer asked for. However, this response is not included in the revised manuscript. I think the NRL change is an important biological insight of this work. Indeed, we have already cited this preprint in our recent review article and discussed this NRL effect there (Bikova et al (2026) Nucleic Acids Res 54, gkag074, https://academic.oup.com/nar/article/54/5/gkag074/8506906). Since the NRL effect is quite central to this work, I think the discussion in response to Reviewer 2 needs to be moved to the main manuscript, and it can be further expanded.

3) As part of the NRL discussion, Reviewer 2 asked to check whether any differences between developmental stages are observed for the DNA fragment size distribution, including dinucleosome sizes. This is an interesting question, and it seems that it has not been addressed in the revised manuscript. To see better the differences in fragment size distributions of different samples, I suggest to modify the Y-axis scale of the figure which you have provided in response to Reviewer 2 (e.g. you can make it log-normal scale, etc) and include this figure in the revised manuscript or supplementary materials. It will be also good to show the original phasogram curves for each developmental stage in a similar overlay format in a separate figure, to be included either in the main manuscript or in the supplementary materials. This will address the concerns of Reviewer 2 regarding biological significance of the NRL results versus MNase-seq digestion variability.

4) In the Methods section, please add technical details of where the Kensche et al data was obtained from (GEO accession number), and how was it processed (e.g. whether separate SRA runs corresponding to the same developmental stage were merged, what was the alignment rate, etc).

5) The Swissknife package needs a reference with names of authors, as listed at their web site: https://fmicompbio.github.io/swissknife/reference/index.html. It can be also noted that this package is using an algorithm that is slightly different from the algorithm in Valouev et al, so it is not a simple implementation of Valouev et al.

6) Line 620 mentions “Custom R script”. Please make this script publicly available.

Best regards,

Vladimir Teif

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Authors:

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Reviewer #1: I thank the authors for their detailed responses and for the substantial revisions made to the manuscript. The revised version is considerably improved, and many of my original concerns have been addressed. In particular, I appreciate the clarification regarding previous reports of +1 nucleosome positioning in Plasmodium falciparum, the improved discussion of methodological limitations, the additional analyses provided throughout the manuscript, and the efforts made to improve reproducibility through the deposition of code, annotations, and outputs in a permanent repository.

Below I provide a point-by-point assessment of the revised manuscript.

1. Clarification of +1 nucleosome positioning in P. falciparum

Assessment: Addressed

The manuscript now appropriately acknowledges previous studies reporting +1 nucleosomes and nucleosome-free regions in P. falciparum. The revised text more accurately defines the novelty of the present study as the identification of phased downstream nucleosome arrays rather than the discovery of the +1 nucleosome itself.

I also appreciate the additional analyses examining the relationship between +1 nucleosome positioning and gene expression, which are consistent with previous observations in the field.

2. Reference nucleosome numbers

Assessment: Addressed

The authors now provide additional context regarding the total number of nucleosomes identified and compare these values with previous studies. This improves the interpretation of the dataset and clarifies how the reference nucleosome set was defined.

3. Use of mono-nucleosome fragments

Assessment: Addressed

The authors clearly explain that the improved nucleosome maps result not only from fragment selection but also from differences in preprocessing and alignment strategies. They also acknowledge that the pipeline has been optimized for mono-nucleosome analysis and that performance on di- and tri-nucleosome fragments remains untested.

I consider this concern adequately addressed.

4. Genome-wide occupancy and chromosomal distribution

Assessment: Largely addressed

The revised manuscript now discusses the accumulation of dynamic nucleosome features at centromeric regions and the depletion observed at chromosome ends. The additional supplementary analyses improve interpretation of these observations and address most of my concerns regarding genome-wide distribution.

5. Dependence on DANPOS

Assessment: Addressed

The relationship between nucDetective and DANPOS is now much clearer. The authors appropriately distinguish between existing tools incorporated into the workflow and the original components developed as part of nucDetective.

6. Reproducibility and data availability

Assessment: Addressed

The addition of the Zenodo repository, together with the clarification regarding scripts, annotations, pipeline outputs, and reproducibility resources, substantially improves transparency and reproducibility.

Remaining concerns

7. Quality control and validation of dynamic nucleosome detection

Assessment: Partially addressed

I appreciate the expanded description of quality-control procedures and the rationale provided by the authors regarding insert-size distributions, TSS profiles, PCA structure, replicate consistency, and visual inspection of nucleosome profiles.

However, my original concern regarding quality control within the Inspector workflow itself remains only partially addressed. The response primarily explains why the authors consider the current workflow sufficient, but additional QC metrics or validation procedures for downstream dynamic nucleosome calls have not been implemented.

I think it is important to distinguish between quality control of MNase-seq data generation and processing, and validation of the biological conclusions derived from dynamic nucleosome detection. While the manuscript now better documents the former, the latter remains less developed.

Similarly, the current strategy of selecting the 20% best-positioned nucleosomes based on fuzziness represents a conservative filtering approach, but it does not directly estimate false-positive rates or independently validate dynamic nucleosome calls. I understand the authors’ argument that no accepted gold-standard dataset exists for this purpose. Nevertheless, I believe the manuscript should more clearly emphasize that the identified loci represent high-confidence candidate dynamic nucleosomes rather than a formally validated set of differential nucleosome events.

8. Statistical support for time-series analyses

Assessment: Partially addressed

The authors now clarify that nucDetective is intended primarily as a screening framework and that dynamic nucleosomes are identified through variance-based ranking rather than formal statistical testing. The addition of a dedicated limitations section is welcome and improves transparency.

However, this clarification also highlights an important limitation of the current approach. Dynamic nucleosomes are not identified using a statistical framework that explicitly models biological variance across conditions. Consequently, the strength of evidence supporting differential nucleosome behaviour remains difficult to assess quantitatively.

I do not necessarily view this as a fatal limitation, particularly given the challenges associated with MNase-seq experiments and the limited number of biological replicates available in many published datasets. However, I recommend that the authors continue to moderate their wording throughout the manuscript and ensure that dynamic nucleosomes are presented within the context of a variance-based prioritization strategy rather than a statistically validated differential analysis.

9. Sequence bias and gDNA normalization

Assessment: Partially addressed

The authors now clearly explain the use of gDNA normalization and provide a rationale for not incorporating sequence normalization into the general workflow. The added discussion improves transparency.

Nevertheless, given the extreme AT-rich composition of the P. falciparum genome and the well-established sequence preferences of MNase, I still consider this an important limitation. The issue is now acknowledged in the Discussion, which is an improvement, but readers should be reminded that the reported nucleosome profiles are not corrected for sequence-specific biases.

10. Statistical support for specific figures

Assessment: Addressed

The corrections regarding figure references, the addition of statistical support for nucleosome repeat length analyses, and the clarification that Figure 5B highlights visual observations rather than statistical significance address my concerns regarding figure interpretation.

Overall assessment

The manuscript has improved substantially and addresses the majority of the concerns raised in my original review. The remaining issues primarily concern validation and interpretation rather than the biological observations themselves.

In particular, I believe the manuscript would benefit from a clearer distinction between: (i) quality control of MNase-seq data processing, (ii) identification of candidate dynamic nucleosomes, and (iii) statistical validation of differential nucleosome behaviour. At present, these concepts occasionally overlap in the discussion of pipeline performance.

Overall, nucDetective appears to be a useful and reproducible framework for identifying and prioritizing candidate dynamic nucleosomes across complex experimental designs. However, I encourage the authors to further moderate statements regarding dynamic nucleosome detection and to emphasize the exploratory nature of the approach where appropriate.

With these revisions, I believe the manuscript would provide a valuable contribution both as a computational resource and as an updated view of chromatin organization in Plasmodium falciparum.

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Reviewer #1: Yes

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Reviewer #1: Yes:  Ibtissam Jabre

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Dear Dr. Längst,

Thank you very much for the revised version of your manuscript. We are pleased to inform you that your manuscript 'Deciphering chromatin architecture and dynamics in Plasmodium falciparum using the nucDetective pipeline' has been provisionally accepted for publication in PLOS Computational Biology.

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Best regards,

Vladimir B Teif, Ph.D.

Academic Editor

PLOS Computational Biology

Shaun Mahony

Section Editor

PLOS Computational Biology