PONE-D-20-11686

Metagenome analysis from the sediment of river Ganga and Yamuna: In search of health beneficial microbiome

PLOS ONE

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Reviewers' comments:

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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: No

Reviewer #2: Yes

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

Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

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: This manuscript by Behera BK et al describes distribution of various beneficial bacterial species in the sediments of two Indian rivers (Ganga and Yamuna) using shutgun metagenomic approach. In this study the authors generated sequencing reads for nine sampling sites; with three sites from three geographically distant regions along the river banks makes the experimental design well thoughtful. They primarily analyzed the sequencing reads using bioinformatics tools and compared average number of species in these three regions. Through system biology based approaches they tried to elucidate the functional relevance of some selected bacteria to aquatic environment.

The study seems to be interesting and well relevant. Although the manuscript depicts to address the problem, it lacks clarity in many aspects and should be addressed before acceptance.

My major concerns are :

1. The criteria used for construction of the beneficial bacteria list is incomprehensible. It looks very strange that beneficial effects of each of these 69 bacteria (Supplementary Table 1) were evidenced by only one study. A simple pubmed search indicates that several of them have pathogenic effect to aquatic organisms. For example, Vibrio mediterranei is associated to major mortality in Pinna nobilis (https://www.sciencedirect.com/science/article/abs/pii/S0044848619324494). Some have role in serious human health hazards. For example consumption of fish infected with V. fluvialis has been reported to cause mild to moderate dehydration, vomiting, fever, abdominal pain and diarrhoea (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2996184/). There are many more such bacteria from this list which has been reported to be associated with serious illness in human and other aquatic organisms. So authors need to define a clear criteria for this classification and provide more than one evidence supporting their health benefit.

2. The abstract describes that a total of 242 health beneficial bacteria were identified. However, rest of the manuscript discusses only 69 (Heatmap figure legend says 67 species). While Kaiju web server performs taxonomic classification of sequencing reads, it is unclear from the manuscript whether the list of beneficial bacteria was created before performing the all data analysis or bacteria names (form Kaiju results) were selected later after a manual literature search?

3. The method section is ambiguous and insufficient to reproduce the results independently. Important analyses such as relative abundance calculation and statistical methods used for significance tests have not been described sufficiently.

4. In many cases the input data are not clear. For example, the inputs for multiple sequence analysis (or multiple sequence alignment???), species Phylogeny construction and PCA are not clear form the text.

5. The purpose of Phylogenetic analysis should be discussed appropriately. The authors conclude that "some of the microbial species are highly conserved throughout the evolution". How come a species can be conserved? Genes/proteins are conserved during evolution of a lineage.

- Bootstrap is a score to show confidence of relatedness or similarily in a clade and does not refer to species closeness.

- Pg 15: What is "evolutionary closed supportive microbiome"??

- Pg 20: What is the difference between "Circular Cladogram" and "Cladogram"?

6. The Relative abundance calculation is totally unclear. In biodiversity studies usually it refers to the number of individuals from i-th speceis divided by total number of individuals from a sample (10.1590/S1516-89132012000200014). It would be useful if the authors provide a concise definition of this index at species level.

7. The GO enrichment and pathway enrichment does not show any relation with heavy metal re-mediation. Mere presence of HMA domain in one protein from one species should not be concluded as "potential heavy metal remediating property". This result should be reported in a subtle way.

8. The rationale behind the STRING protein-protein interaction is not clear.

9. In introduction, the manuscript describes that there are many studies on discovering microbiome from natural streams of other countries. However, in the discussion the results obtained from present study were not compared enough with results from past metagenomic studies. The discussion should put the results in a more broader context.

10. The conclusion that "sediment metagenome of the river Ganga and Yamuna manifests the enriched microbial distribution of health beneficiary microbes" seems biased. Since the study analyses pre-selected list of 69 species, whether the samples are enriched in beneficial bacteria or not is difficult to conclude.

## Minor Comments

1. Lack of page and line numbers in the manuscript makes it hard to review. I have tried to write the exact phrase so that the authors can find the concerned lines.

2. Reference-1 seems bit out of context. Also the list of authors seems incomplete (Compare with this https://www.scienceopen.com/document?vid=b20de2d9-e8dd-478e-8e2b-5fb9d967b6ea)

3. Pg -12 "of health beneficialmicrobiome" should be "beneficial microbiome"

4. Substitution models and number of bootstrap replicate number should be mentioned in a phylogenetic analysis.

5. For all software tools please provide appropriate citation. Citations help the labs to receive funding and maintain the tool.

6. Pg-12: "each sample were then assembled into scaffolds using CLC". Please provide the number of scaffolds and their average length. There is no information about these scaffolds in the results.

7. Pg 14: Please fix the scientific names: Bacillus Clausii, Enterococcus Hirae, Leuconostocme senteroides, Pediococcus Acidilactici,

Pseudomonas Stutzeri, Roseobacter Litoralis

8. Pg-14: "four Roseobacter (R. litoralis, R. denitrificans, R. Litoralis and R. denitrificans)"- R. litoralis and R. Litoralis are similar or diffenent. It makes the list fof species to 68.

9. Multiple species has Similar genus abbreviation. Consider using two letter abbreviation; for example Vi. mediterranei and Va. fluvialis bH819.

10. "Lactococcuslactis " should be Lactococcus lactis ???

11. "Leuconostocme senteroides" should be Leuconostoc mesenteroides ???

12. "boot strap" or bootstrap ???

13. Pg 15: Heatmap is a way of data representation. What is Heatmap analysis??

14. Pg 15: "beneficialspecies in the" should be "beneficial species in the" ??

15. The term "significantly" should be used more cautiously. It should be used only for statistical tests and the alpha (p-value) must be specified.

16. The term "a large number of" seems very vague. Rather, shuch numbers should be descried in a quantitative manner (in terms of percentage).

17. Pg 35: I would suggest to replace Table 2 with a box-plot. It would reduce the number of pages while making the results more informative and attractive.

18. The bootstrap values should be displayed on the tree in appropriate way.

My intention behind extensive comments should be taken in a constructive manner to improve the manuscript.

Thank you.

Reviewer #2: In the manuscript PONE-D-20-11686 authors have performed metagenome analysis to identify microbes with favorable health outcome to humans as well as all other strata of organisms present in the trophic pyramid. Through contemporary high throughput method authors have reported presence of Lactobacillus, at Farakka stretch of river Ganga and at New Delhi stretches of river Yamuna, whereas Roseobacter spp. was found to be highly enriched at Kanpur sites of river Ganga. Such finding would definitely helpful in isolating beneficial microbes from river sediments for future industrial application. The article meets scientific standard for publication.

However, authors may carry out minor revision to improve quality of the manuscript.

1. In method logic behind setting up string analysis parameter could be mentioned.

2. Authors could mention limitations and future direction of the study in the conclusion.

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Reviewer #1: Yes: Kanhu Charan Moharana

Reviewer #2: Yes: Dr. Dibyabhaba Pradhan

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Metagenome analysis from the sediment of river Ganga and Yamuna: In search of beneficial microbiome

PONE-D-20-11686R1

Dear Dr. Behera,

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,

Manas Ranjan Dikhit

Academic Editor

PLOS ONE

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