Whole blood transcriptomics analysis of Indonesians reveals translocated and pathogenic microbiota in blood

Katalina Bobowik; Muhamad Fachrul; Chelzie Crenna Darusallam; Pradiptajati Kusuma; Herawati Sudoyo; Clarissa A. Febinia; Safarina G. Malik; Christine A. Wells; Irene Gallego Romero

doi:10.1371/journal.pone.0328788

Peer Review History

Original SubmissionJune 24, 2024
24 Jun 2024 Author Response Attachments Attachment Submitted filename: 126657_1_rebuttal_3051114_sf265p.docx https://doi.org/10.1371/journal.pone.0328788.r001
29 Oct 2024 Decision Letter - Rajesh Singh Rathore, Editor PONE-D-24-24494The whole blood microbiome of Indonesians reveals translocated and pathogenic microbiotaPLOS ONE Dear Dr. Irene Gallego Romero 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 13 2024 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. 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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 update your submission to use the PLOS LaTeX template. The template and more information on our requirements for LaTeX submissions can be found at http://journals.plos.org/plosone/s/latex. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: “We would like to acknowledge all of the study participants who generously consented to genome sequencing in the original study, as well as Emily R. Davenport, Murray P. Cox and members of the Gallego Romerogroup for helpful comments on the manuscript. St Vincent’s Institute acknowledges the infrastructuresupport it receives from the National Health and Medical Research Council Independent Research InstitutesInfrastructure Support Program and from the Victorian Government through its Operational Infrastruc ture Support Program. PK is supported by the Wellcome Trust International Training Fellowship (no.222992/Z/21/Z)” We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: “The authors received no specific funding for this work.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 4. We notice that your supplementary figures are included in the manuscript file. Please remove them and upload them with the file type 'Supporting Information'. Please ensure that each Supporting Information file has a legend listed in the manuscript after the references list. 5. Please upload a copy of Supporting Information Figure/Table/etc. Supplementary tables 1 to 8 which you refer to in your text on pages 26 and 27. Additional Editor Comments: The article titled "The Whole Blood Microbiome of Indonesians Reveals Translocated and Pathogenic Microbiota" describes the use of transcriptomics data to investigate the presence of pathogens in whole blood samples specifically in Indonesian region. The authors have addressed prior reviewer enquiries; still, major minor remarks are included as per other reviewer and some are as below: a) Please explain how the author relates this study's impact to John P. Hanley et al. (2021) and Louis et al. (2005). What is the significance of whole blood transcriptome analysis, which identified distinct gene sets that correlated with viremia and revealed varying gene expression patterns before and after infection, implying immune state changes? b) What are the hematological changes and cellular composition changes and Gene Expression Patterns in relational to host and pathogen? c) Why did the author choose an RNA-based investigation while prior research using mNGS and shotgun metagenomics demonstrated a more extensive analysis? d) Author mentioned “Recent studies have found no evidence of a core microbiome circulating in the blood of healthy Stanley plot individuals” and “However, not enough studies have been done to confirm whether this lack of a core microbiome is also consistent in regions where infectious diseases are endemic” but plethora of literature is available depicting role of core microbiome circulating in the blood of healthy individuals – e) Study by Païssé et al. (2016), Study by Castillo et al. (2019), Blauwkamp et al….. so on, Nature study by Ling et al. (2023) - large-scale study analyzed blood samples from 9,770 healthy individuals. f) Why does the author focus exclusively on Indonesia—specifically Mentawai, Sumba, and the Indonesian portion of New Guinea Island—as the data set for this study? g) Enhance the methodology section by providing a detailed description that emphasizes sample collection, RNA isolation procedures, library preparation, quantification, and host DNA depletion techniques. Include the catalogue information for the kits used to facilitate replication by others, as well as the parameters employed for the Illumina HiSeq 2500, and ensure appropriate citations are included. h) Provide the consent information for all human individuals (Weight, Hight, BMI etc). i) Provide the mapped and unmapped paired-end data separately? j) Please verify that the given data files encompass all individual samples in the repository. k) Instead of a circular bar plot, the author should present a stanley plot that shows the interconnected changes between the samples; this will provide a better understanding of the data. l) For compositional data analysis, the author must execute isometric log-ratio (ILR) transformation, which preserves all metric aspects of the data, coordinates with a non-singular covariance matrix, and allows for simpler interpretation of results. Alternatively, utilise the Robust Centred Log-Ratio (rCLR) and Inter-Quartile Log-Ratio (IQLR) Transformations. m) The justification for doing Welch's t-test in this investigation is unclear author should use Limma-Voom, NOIseq, SAMseq, etc. for analysis? n) In discussion part author mentioned “Our understanding of pathogens found within remote regions of Indonesia, along with their impact on gene expression, is limited. But several published studies on global scenario showed impact of pathogens are associated with environmental factors like temperature, humidity, location of longitude Natri et al., 2020 etc. As author used European data in this study but some studies mentioned the commonalities in gene regulation between Indonesia and Europe (Natri wt al., 2022), So, instead of using European data, I strongly recommend that the author conduct a comparative and conclusive study using data from other continents. [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: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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: 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 ****** 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 titled “The Whole Blood Microbiome of Indonesians Reveals Translocated and Pathogenic Microbiota” presents the use of transcriptomics data to explore the presence of pathogens in whole blood samples. The authors have responded to previous reviewer questions; however, a few minor comments are provided below: 1. If the authors agree, the title can be revised to "Whole Blood Transcriptomics Analysis of Indonesians…" to provide a clearer picture of the study. 2. If the primary aim of the study is to detect pathogens, a microbial-targeted metagenomic study could be considered. This approach may help address the issue of contaminated human reads that the authors encountered. Are there any previous studies that used metagenomic analysis for pathogen detection, and did they produce similar or different results? If using RNA-seq is more critical than metagenomics in this context, the authors could clarify this in the introduction or discussion section. Reviewer #2: The manuscript under review focuses on a relevant but controversial topic: Assessing the state of the blood microbiome. I would like to focus on the ambiguity of the task that the authors set in their study. On the one hand, the presence of genome fragments of certain types of microorganisms in the blood was confirmed by experimental studies. On the other hand, the functional role of some of them remains unclear, especially in the context of the blood microbiome of healthy individuals. Although there is a point of view that there are no absolutely healthy people, the genomes of individuals are noted to be carriers of various potential pathological structures. Note that the authors used adequate methods for both obtaining data sets and bioinformatic and statistical processing to prove their hypothesis. In general, this manuscript is recommended for publication. I offer several technical recommendations: 1. The authors did not provide additional tables; therefore, their quality and necessity were not assessed. 2. Methods, Datasets section, 1 paragraph: It is necessary to provide a reference to the citation in the text: "In the original Natri et al. study…». 3. Methods, Datasets section, paragraph 2: It is necessary to agree with the list of references and correct the author’s last name “The first dataset comes from Tran et al. [28, 29] …”. ****** 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: No Reviewer #2: 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. https://doi.org/10.1371/journal.pone.0328788.r002
Revision 1
2 May 2025 Author Response We have uploaded an updated document (response_to_editor_reviewers_PLOSONE_EiC_02052025.docx) addressing all the point-by-point comments from the editor and reviewers. The response is also detailed below: 1. Point-by-point description of the revisions Editor: a) Please explain how the author relates this study's impact to John P. Hanley et al. (2021) and Louis et al. (2005). What is the significance of whole blood transcriptome analysis, which identified distinct gene sets that correlated with viremia and revealed varying gene expression patterns before and after infection, implying immune state changes? Our response: We are not sure which exact papers the editor referred to, but the comment is not within the immediate scope of our manuscript as our study contains only pre-infectious individuals. b) What are the hematological changes and cellular composition changes and Gene Expression Patterns in relational to host and pathogen? Our response: Our previous works with this dataset have covered this, and we have cited them (references 25-27; https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1008749, https://www.sciencedirect.com/science/article/pii/S0002929721004377, https://www.biorxiv.org/content/10.1101/2021.01.07.425684v1). In short, we found no significant difference in gene expression levels or cellular composition associated with pathogen loads in our cohort. This is not surprising as our cohort is comprised of self-reported healthy individuals, so even those with detectable malaria loads were not presenting symptoms. c) Why did the author choose an RNA-based investigation while prior research using mNGS and shotgun metagenomics demonstrated a more extensive analysis? Our response: We have mentioned in our abstract and discussion sections how this approach is not a one-to-one replacement for shotgun metagenomics as a main surveillance tool, but rather a valuable retrospective tool to look at transcriptomic data generated in the past with no matched metagenomic data available. d) Author mentioned “Recent studies have found no evidence of a core microbiome circulating in the blood of healthy Stanley plot individuals” and “However, not enough studies have been done to confirm whether this lack of a core microbiome is also consistent in regions where infectious diseases are endemic” but plethora of literature is available depicting role of core microbiome circulating in the blood of healthy individuals – e) Study by Païssé et al. (2016), Study by Castillo et al. (2019), Blauwkamp et al….. so on, Nature study by Ling et al. (2023) - large-scale study analyzed blood samples from 9,770 healthy individuals. Our response: The consensus in the field is clearly converging on there being no core blood microbiome. We believe the Ling et al study mentioned by the editor is actually a reference to Tan et al. (2023) (https://www.nature.com/articles/s41564-023-01350-w), which emphasize the notion that the blood of healthy individuals do not support a consistent core microbial community. We cited this paper in our manuscript as reference number 19 (page 14, first paragraph of discussion in our manuscript) as it supports our argument. We also cited Castillo et al. (2019) as reference number 48, in which the paper presents the evidence of eukaryotes and viruses in blood transcriptome yet also challenges their presence in healthy human blood, not concluding their definite existence and encouraging researchers to be more careful with experimental design (which also supports our main argument). This topic in the microbiome / metagenomics field has been, and remains highly debated, with new evidence challenging the accepted paradigm; the Païssé et al. (2016) reference the editor mentioned is already 8 years old and more recent and robust evidence (like the Tan et al. paper) have come out. We’re unsure which Blauwkamp et al. paper the editor referred to as he did not specify the year, but the one in 2019 (https://www.nature.com/articles/s41564-018-0349-6) is not relevant to the author’s point in depicting circulating microbiome in healthy individuals. f) Why does the author focus exclusively on Indonesia—specifically Mentawai, Sumba, and the Indonesian portion of New Guinea Island—as the data set for this study? Our response: We have mentioned in the abstract and introduction how Indonesia is such an underrepresented population with high numbers of endemic and emerging infectious diseases, and why we specifically chose those three regions (These populations span a gradient from west to east across Indonesia, thus capturing pathogens along the main geographical axis of the country). There seems to be a fundamental misunderstanding of our manuscript by the editor, and a re-read or a different editor may help clarify this point. g) Enhance the methodology section by providing a detailed description that emphasizes sample collection, RNA isolation procedures, library preparation, quantification, and host DNA depletion techniques. Include the catalogue information for the kits used to facilitate replication by others, as well as the parameters employed for the Illumina HiSeq 2500, and ensure appropriate citations are included. Our response: This was already presented and provided in the original paper (which we’ve cited). It is not a new dataset and therefore would be weird to provide them again here. h) Provide the consent information for all human individuals (Weight, Hight, BMI etc). Our response: Consent is not related to anthropometric descriptors. Regardless, we’ve always cited the original paper the data was first presented in. i) Provide the mapped and unmapped paired-end data separately? Our response: This was already presented and provided in the original paper (which we’ve cited). It is not a new dataset and therefore would be weird to provide them again here. j) Please verify that the given data files encompass all individual samples in the repository. Our response: This was already presented and provided in the original paper (which we’ve cited). It is not a new dataset and therefore would be unfit to provide them again here. k) Instead of a circular bar plot, the author should present a stanley plot that shows the interconnected changes between the samples; this will provide a better understanding of the data. Our response: The editor is very unclear in their demands. There is no such widely acceptable type of plotting style as a “Stanley plot”. We simply do not understand what the editor meant by this. If the editor invented this type of plots, we are not aware. l) For compositional data analysis, the author must execute isometric log-ratio (ILR) transformation, which preserves all metric aspects of the data, coordinates with a non-singular covariance matrix, and allows for simpler interpretation of results. Alternatively, utilise the Robust Centred Log-Ratio (rCLR) and Inter-Quartile Log-Ratio (IQLR) Transformations. Our response: We have used the Centred Log-Ratio (CLR) transformation for our data, which is adequate for compositional data, as we have explained in the methods section (page 7, Sample clustering subsection). m) The justification for doing Welch's t-test in this investigation is unclear author should use Limma-Voom, NOIseq, SAMseq, etc. for analysis? Our response: The methods the editor suggested are not recommended for microbiome data. As explained in our method section, we used ALDEx2 to test for differences in species composition between populations, which applies CLR-transformation to correct for uneven library depth and data compositionality and uses Welch’s t-test for significance test (page 7, Differential abundance testing and diversity estimation subsection). We ran Welch’s t-test and default 128 Monte Carlo simulations for the phylum-level analyses, as ALDEx2 is more suited for lower-level classification (species, genus) differential abundance. n) In discussion part author mentioned “Our understanding of pathogens found within remote regions of Indonesia, along with their impact on gene expression, is limited. But several published studies on global scenario showed impact of pathogens are associated with environmental factors like temperature, humidity, location of longitude Natri et al., 2020 etc. As author used European data in this study but some studies mentioned the commonalities in gene regulation between Indonesia and Europe (Natri wt al., 2022), So, instead of using European data, I strongly recommend that the author conduct a comparative and conclusive study using data from other continents. Our response: We also included an African cohort (Mali) for validation, which resembles the endemic / rural environment of our Korowai donors more. Again, there seems to be a fundamental misunderstanding of our manuscript by the editor, and a re-read or a different editor may help clarify this point. Reviewer 1: The manuscript titled “The Whole Blood Microbiome of Indonesians Reveals Translocated and Pathogenic Microbiota” presents the use of transcriptomics data to explore the presence of pathogens in whole blood samples. The authors have responded to previous reviewer questions; however, a few minor comments are provided below: 1. If the authors agree, the title can be revised to "Whole Blood Transcriptomics Analysis of Indonesians…" to provide a clearer picture of the study. Our response: We thank Reviewer 1 for the suggestion and have amended it. 2. If the primary aim of the study is to detect pathogens, a microbial-targeted metagenomic study could be considered. This approach may help address the issue of contaminated human reads that the authors encountered. Are there any previous studies that used metagenomic analysis for pathogen detection, and did they produce similar or different results? If using RNA-seq is more critical than metagenomics in this context, the authors could clarify this in the introduction or discussion section. Our response: As mentioned in our abstract and discussion sections, our approach is more suited as a retrospective surveillance tool in cases where only past transcriptomic data are available with no matched metagenomic data or fresh samples, not as a main disease surveillance tool. We have cited a few studies using microbial-targeted metagenomic approaches for pathogens and mentioned them in the introduction, such as the study by Kafetzopoulou et al. (2019) who used Nanopore sequencing for Lassa virus (https://pmc.ncbi.nlm.nih.gov/articles/PMC6855379/). Reviewer 2: The manuscript under review focuses on a relevant but controversial topic: Assessing the state of the blood microbiome. I would like to focus on the ambiguity of the task that the authors set in their study. On the one hand, the presence of genome fragments of certain types of microorganisms in the blood was confirmed by experimental studies. On the other hand, the functional role of some of them remains unclear, especially in the context of the blood microbiome of healthy individuals. Although there is a point of view that there are no absolutely healthy people, the genomes of individuals are noted to be carriers of various potential pathological structures. Note that the authors used adequate methods for both obtaining data sets and bioinformatic and statistical processing to prove their hypothesis. In general, this manuscript is recommended for publication. I offer several technical recommendations: Our response: We thank Reviewer 2 for their fair assessment. 1. The authors did not provide additional tables; therefore, their quality and necessity were not assessed. Our response: We have provided 8 supplementary tables containing the read depth quality and differential abundance results of all the involved datasets. We understand if Reviewer 2 might not have received the supplementary files during the review process. 2. Methods, Datasets section, 1 paragraph: It is necessary to provide a reference to the citation in the text: "In the original Natri et al. study…». Our response: We thank Reviewer 2 for pointing this out and have amended it. 3. Methods, Datasets section, paragraph 2: It is necessary to agree with the list of references and correct the author’s last name “The first dataset comes from Tran et al. [28, 29] …”. Our response: We thank Reviewer 2 for pointing this out and have confirmed the author’s name is correct (Tuan M. Tran, hence Tran et al.). Attachments Attachment Submitted filename: response_to_editor_reviewers_PLOSONE_EiC_02052025.docx https://doi.org/10.1371/journal.pone.0328788.r003
27 Jun 2025 Decision Letter - Sylvia Maria Bruisten, Editor PONE-D-24-24494R1Whole blood transcriptomics analysis of Indonesians reveals translocated and pathogenic microbiota in bloodPLOS ONE Dear Dr. Gallego Romero, 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 find my comments as new editor/ reviwer 3 below. Please submit your revised manuscript by Aug 11 2025 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, Sylvia Maria Bruisten, 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. Additional Editor Comments: This manuscript was previously commented on by another editor and two reviewers. The two reviewers had minor comments which were well answered in this first Rebuttal version. The comments from the editor met with difficulties for the authors to answer since there seem to be misunderstandings on what this manuscript aims to describe. As the new scientific editor I think that this is a well written and mostly comprehensive manuscript that describes interesting data on transcriptome (RNA seq) data on previously collected whole blood samples from 3 Indonesian populations. These data were compared to similar data from a population in Mali and a small population from London, UK. I have one major and some minor comments to further improve the manuscript. Major comment As was also mentioned earlier by reviewer 2, Supplementary Tables 1 to 8 cannot be found in the document submitted to PlosOne (PONE-D-24-24494R1). Please provide these Suplementary Tables with the next Rebuttal version. Minor comments Abstract It is mentioned that ‘two pathogens—Flaviviridae and Plasmodium—are the most predominantly abundant’. I suggest to change ‘pathogens’ into ‘taxa’ since that is more accurate. If the authors like they can add that these taxa includes pathogens such as Dengue viruses and malaria causing parasites. In addition there are 5 ‘key highlights’ mentioned in the letter to the editor from May 2025. The first three key points deserve to be also mentioned in the abstract because in my view this is what the reader wants to know. There is no limitation in the number of words for an abstract for PlosOne, so this can be formulated it as comprehensive as needed. Results Supplemental Figure 1 and Figure 2: The legend colours and text do not everywhere line up; please adjust so these are aligned properly. For example ‘Chordata’ in Suppl Fig 1 is now next to a blue block whereas it should be next to a purple one. And ‘KOR’ should be next to the red blocks. Supplementary Figure 5 contains interesting data which I think deserve to be shown in the main text. Please consider moving it there. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: No extra comments, reviewers were not again invited since their pints were properly addressed. [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. Attachments Attachment Submitted filename: Comments_Bobowik_etal_PONE-D-24-24494-R1-June 2025.doc https://doi.org/10.1371/journal.pone.0328788.r004
Revision 2
2 Jul 2025 Author Response Major comment As was also mentioned earlier by reviewer 2, Supplementary Tables 1 to 8 cannot be found in the document submitted to PlosOne (PONE-D-24-24494R1). Please provide these Suplementary Tables with the next Rebuttal version. Our answer: We thank the editor for pointing this out and have now included supplementary tables 1-8 in the new version (as two separate excel documents). Minor comments Abstract It is mentioned that ‘two pathogens—Flaviviridae and Plasmodium—are the most predominantly abundant’. I suggest to change ‘pathogens’ into ‘taxa’ since that is more accurate. If the authors like they can add that these taxa includes pathogens such as Dengue viruses and malaria causing parasites. Our answer: We thank the editor for the suggestion and have amended the section. In addition there are 5 ‘key highlights’ mentioned in the letter to the editor from May 2025. The first three key points deserve to be also mentioned in the abstract because in my view this is what the reader wants to know. There is no limitation in the number of words for an abstract for PlosOne, so this can be formulated it as comprehensive as needed. Our answer: We thank the editor for the suggestion and have incorporated the first three key points into the abstract. Results Supplemental Figure 1 and Figure 2: The legend colours and text do not everywhere line up; please adjust so these are aligned properly. For example ‘Chordata’ in Suppl Fig 1 is now next to a blue block whereas it should be next to a purple one. And ‘KOR’ should be next to the red blocks. Our answer: We thank the editor for the observation. It seems that the tracked conversion from our Latex pdf output to a word document has caused the individual elements of the figures to be individualized instead of merged as one, as the figures look fine on pdf. We have now amended this. Supplementary Figure 5 contains interesting data which I think deserve to be shown in the main text. Please consider moving it there. Our answer: We thank the editor for the suggestion and have moved Supplementary Figure 5 into the main text as Figure 3. Attachments Attachment Submitted filename: response_to_editor_June2025.doc https://doi.org/10.1371/journal.pone.0328788.r005
8 Jul 2025 Decision Letter - Sylvia Maria Bruisten, Editor Whole blood transcriptomics analysis of Indonesians reveals translocated and pathogenic microbiota in blood PONE-D-24-24494R2 Dear Dr. Gallego Romero, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Sylvia Maria Bruisten, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): All comments were taken into consideration and all points were adjusted to satisfaction. Reviewers' comments: https://doi.org/10.1371/journal.pone.0328788.r006
Formally Accepted
Acceptance Letter - Sylvia Maria Bruisten, Editor PONE-D-24-24494R2 PLOS ONE Dear Dr. Gallego Romero, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Sylvia Maria Bruisten Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0328788.r007

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