Reviewer ’s report  
 
Title: In search for interplay between stool microRNAs, mi crobiota  and short chain fatty acids in 
Crohn’s disease. A preliminary study.  
 
Version: 0 Date: 07 Apr 2020  
 
Reviewer: Alexander V. Tyakht  
 
Reviewer's report:  
 
Grammar and language generally should be revised: some microbial taxa names are misspelled, 
punctuation signs missing, fragmented sentences, etc. Some are listed below.  
 
 
 
Last sentence in Abstract Background is fragmented.  
 
16s rRNA -> 16S rRNA (here and elsewhere)  
 
A. Results: It is not that the "taxa differed" rather that their relative abundance levels were 
different between the groups, isn't it? Consider rephrasing.  
 
Main text.  
 
41: 43 -45: Why is there an antithesis (" Although")? The two parts do not appear to contradict.  
 
49: Consider punctuation: "(IBDs) - Crohn's disease (CD) and ulcerative colitis (UC)".  
 
55: GaMmaproteobacteria  
 
57: FAecalibacterium prausnitziI  
 
59: 90% of the phylogenetic diversity? of the total m icrobial number? Please specify.  
Also, members of other phyla (e.g. Actinobacteria) can also produce SCFAs - consider not 
excluding them here.  
 
61: Dietary fibers are not the sole precursors of SCFA in the gut - at least another one being 
amino -acids resul ting from protein breakdown, perhaps to a lower extent: e.g. see 
https://sfamjournals.onlinelibrary.wiley.com/doi/pdf/10.1111/1462 -2920.13589 .  
 
67: Another linked reason worth mentioning is reduced fiber consumption in acute IBD patients. 
Consider includi ng, if possible, with reference.  
 
81: At which hospital were the patients enrolled and in which year? Is information about ethnic 
group available? (as genetics are an important factor contributing to CD risk)  
 
 
83: There is the first time bacterial mRNA ar e mentioned. If the bacterial gene expression has 
been conducted, it is an important expansion to the multi -omics study design, consider 
mentioning it in the Abstract, amending Introduction respectively.  
 
94: Please give more details on healthy controls as sessment. If it was only self -reported, without 
examination, did they at least complete Rome criteria or other well -being/gastrointestinal 
questionnaire?  
 
108: Consider listing the variable regions of the gene targeted by the kit.  
 
109: Perhaps, it is not that the bam files were unmapped in the sense of being unclassified - 
rather just "raw bam files"?  
Consider capitalizing fastq and fasta everywhere.  
 
125: If available, the data on Bristol stool scale should be included. At least, i f there were there 
any extreme values (diarrhea or very solid stool), I wonder how it affected the normalization of 
miRNA levels.  
 
166: It is suggested that the 16S rRNA microbiome survey term is used instead of metagenomics 
(the latter recommended to be r eserved for "shotgun" analysis only).  
 
167: Is it that the coverage was higher than targeted read count in all cases or there were >92000 
high-quality reads per sample?  
 
168: Consider changing: in more than 0.1% of reads -> at levels >0.1%  
 
173: I s it that Enterobacteriaceae was overrepresented in controls? Please reformulate.  
Did the method allow to distinguish Escherichia from Shigella based on 16S rRNA reads, as 
appears from the text? if not, consider changing to Escherichia/Shigella.  
 
189: What  does the genus name Prevotella_2 refer to? Perhaps, it is a notation from an older 
version of SILVA database - please check the latest release that has many of such issues 
resolved.  
 
201: Please explain the rationale behind this threshold. Can it be sugge sted from a 
distribution/histogram analysis as a separator between noise - and signal -level detection or 
elsewise?  
 
208: At least for these significant miRNA matches, please provide their length to orient the 
reader.  
 
Overall, could the authors provide more  interpretation related to the miRNAs associated with the 
disease? For example, is it possible to conduct some sort of enrichment analysis of them to look 
for functional explanation of these differences? Additional approach could be literature search if 
gut levels of them have been linked to this or other diseases.  
 
256-261: During all -to-all correlation analysis of multiple metabolites, miRNAs and microbial 
taxa, multiple comparison adjustment should have been performed. Please ensure that it has been 
done (does not appear from the Supplementary Table S6 at least). Considering significance, an 
adjusted p -value threshold should be examined, not correlation coefficient (apparently, threshold 
is > 0.6 here).  
 
269-270: "Opportunistic pathogens can also init iate IBD development through a process 
involving gut dysbiosis" - the reference does not support the statement: it just links acute enteric 
bacterial infection with chronic inflammatory disease, not IBD. Consider rephrasing or finding a 
more supporting ref erence.  
 
272: not their microbiomes but the microbiomes of patients with the respective diseases.  
 
278-279: Could the authors comment on the interesting fact that active CD is characterized with 
higher alpha -diversity than the inactive form? (despite the fact that inactive CD is clinically more 
resembling normal state). Also, the SCFA levels behave simi larly.  
 
279: Please revise: 16S rRNA sequencing does not allow to get data on amounts - just relative 
abundance.  
 
280-282: Consider a more specific and lighter statement instead of "aggressive":  the gut 
representatives of the phylum Bacteroidetes are most ly major commensal microbes.  
In the statement about butyrate, the fact itself is worth double -checking (Firmicutes are more 
capable in terms of butyrate, e.g. see https://mbio.asm.org/content/5/2/e00889 -14.abstract ), as 
well as the reference is not the mo st relevant.  
 
283: The reference [55] is not a review but a research study. Consider citing a more applicable 
meta -analysis/review of IBD -microbiome studies here.  
 
287: "Faecalibacterium, Oscillospira, and Feacalibacterium prausnitzii" - the list appear 
redundant - the first and the last items.  
 
283-338: As the Discussion section is too large, consider compacting this part containing mostly 
literature review with few direct links to the present findings.  
 
321: Consider choosing a consistent single term for each SCFA throughout the manuscript: e.g. 
butanoic acid and not butyrate, etc.  
 
348: Is there existing evidence of reverse: that host modulates microbiome via miRNA pool  
alterations?  
 
352: As in the case of microbes, it is the levels of miRNAs that were d ifferent between the 
groups, not the miRNAs themselves, isn't it?  
 
363: "inter -species" - is it about host -microbe, not microbe -microbe interaction?  
 
385: As far as the reviewer is concerned, correlations cannot have roles; it is just the reflection of 
the mechanisms with hidden causal links.  
 
 
 
The link to deposited metagenomic data is not valid (apparently, yet to be made public?).  
 
Figure 1: The figure is not very illustrative. Consider limiting to major OTUs, adding their 
taxonomic names, ordering the b arplots in a meaningful way. Besides, the colored frames 
(notation of "Phylum") are not visible, at least provided the image quality.  
 
Figure 2,3: consider keeping the same order of groups for boxplots (not to confuse the reader).  
 
Figure 5: Words are unreadable, together with low image quality and many layers deeming the 
whole picture incomprehensible. Should it be circular not oval also?  
 
Suppl Figure 1: consider arranging the plots in 3 x 2 grid.  
 
Suppl table 1: "baseMean - mean n ormalized count in all samples in experiment" - how was it 
calculated? Could it be dependent on read coverage? was rarefaction performed beforehand?  
There are multiple misprints "statistic", "p -vaue" in suppl. tables.  
 
Are the methods appropriate and well described?  
If not, plea se specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the dat a shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what as pects require further 
assessment in your comments to the editors.  
 
I am able to assess the statistics  
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of  competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1.  Have you in the past five years received reimbursements, f ees, funding, or salary 
from an organisation that may in any way gain or lose financial ly from the 
publication of this manuscript, either now or in the future?  
 
2.  Do you hold any stocks or shares in an organisation t hat may in any way gain or 
lose financially from the publication of this  manuscript, either now or in the 
future?  
 
3.  Do you hold or are you currently applying for any patents  relating to the content 
of the manuscript?  
 
4.  Have you received reimbursements, fees, funding, or salary from an organization 
that holds or has applied f or patents relating to the content of the manuscript?  
 
5.  Do you have any other financial competing interests?  
 
6.  Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have  no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests.  
 
I agree to the open peer review policy of the journal. I understan d that my name will be included 
on my report to the authors and, if the manuscript is accepted f or publication, my named report 
including any attachments I upload will be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be included in my named report can be in cluded as confidential comments 
to the editors, which will not be published.  
 
I agree to the open pee r review policy of the journal  
Reviewer ’s report  
 
Title: In search for interplay between stool microRNAs, mi crobiota  and short chain fatty acids in 
Crohn’s disease. A preliminary study.  
 
Version: 0 Date: 07 Apr 2020  
 
Reviewer: Alexander V. Tyakht  
 
Reviewer's report:  
 
Grammar and language generally should be revised: some microbial taxa names are misspelled, 
punctuation signs missing, fragmented sentences, etc. Some are listed below.  
 
 
 
Last sentence in Abstract Background is fragmented.  
 
16s rRNA -> 16S rRNA (here and elsewhere)  
 
A. Results: It is not that the "taxa differed" rather that their relative abundance levels were 
different between the groups, isn't it? Consider rephrasing.  
 
Main text.  
 
41: 43 -45: Why is there an antithesis (" Although")? The two parts do not appear to contradict.  
 
49: Consider punctuation: "(IBDs) - Crohn's disease (CD) and ulcerative colitis (UC)".  
 
55: GaMmaproteobacteria  
 
57: FAecalibacterium prausnitziI  
 
59: 90% of the phylogenetic diversity? of the total m icrobial number? Please specify.  
Also, members of other phyla (e.g. Actinobacteria) can also produce SCFAs - consider not 
excluding them here.  
 
61: Dietary fibers are not the sole precursors of SCFA in the gut - at least another one being 
amino -acids resul ting from protein breakdown, perhaps to a lower extent: e.g. see 
https://sfamjournals.onlinelibrary.wiley.com/doi/pdf/10.1111/1462 -2920.13589 .  
 
67: Another linked reason worth mentioning is reduced fiber consumption in acute IBD patients. 
Consider includi ng, if possible, with reference.  
 
81: At which hospital were the patients enrolled and in which year? Is information about ethnic 
group available? (as genetics are an important factor contributing to CD risk)  
 
 
83: There is the first time bacterial mRNA ar e mentioned. If the bacterial gene expression has 
been conducted, it is an important expansion to the multi -omics study design, consider 
mentioning it in the Abstract, amending Introduction respectively.  
 
94: Please give more details on healthy controls as sessment. If it was only self -reported, without 
examination, did they at least complete Rome criteria or other well -being/gastrointestinal 
questionnaire?  
 
108: Consider listing the variable regions of the gene targeted by the kit.  
 
109: Perhaps, it is not that the bam files were unmapped in the sense of being unclassified - 
rather just "raw bam files"?  
Consider capitalizing fastq and fasta everywhere.  
 
125: If available, the data on Bristol stool scale should be included. At least, i f there were there 
any extreme values (diarrhea or very solid stool), I wonder how it affected the normalization of 
miRNA levels.  
 
166: It is suggested that the 16S rRNA microbiome survey term is used instead of metagenomics 
(the latter recommended to be r eserved for "shotgun" analysis only).  
 
167: Is it that the coverage was higher than targeted read count in all cases or there were >92000 
high-quality reads per sample?  
 
168: Consider changing: in more than 0.1% of reads -> at levels >0.1%  
 
173: I s it that Enterobacteriaceae was overrepresented in controls? Please reformulate.  
Did the method allow to distinguish Escherichia from Shigella based on 16S rRNA reads, as 
appears from the text? if not, consider changing to Escherichia/Shigella.  
 
189: What  does the genus name Prevotella_2 refer to? Perhaps, it is a notation from an older 
version of SILVA database - please check the latest release that has many of such issues 
resolved.  
 
201: Please explain the rationale behind this threshold. Can it be sugge sted from a 
distribution/histogram analysis as a separator between noise - and signal -level detection or 
elsewise?  
 
208: At least for these significant miRNA matches, please provide their length to orient the 
reader.  
 
Overall, could the authors provide more  interpretation related to the miRNAs associated with the 
disease? For example, is it possible to conduct some sort of enrichment analysis of them to look 
for functional explanation of these differences? Additional approach could be literature search if 
gut levels of them have been linked to this or other diseases.  
 
256-261: During all -to-all correlation analysis of multiple metabolites, miRNAs and microbial 
taxa, multiple comparison adjustment should have been performed. Please ensure that it has been 
done (does not appear from the Supplementary Table S6 at least). Considering significance, an 
adjusted p -value threshold should be examined, not correlation coefficient (apparently, threshold 
is > 0.6 here).  
 
269-270: "Opportunistic pathogens can also init iate IBD development through a process 
involving gut dysbiosis" - the reference does not support the statement: it just links acute enteric 
bacterial infection with chronic inflammatory disease, not IBD. Consider rephrasing or finding a 
more supporting ref erence.  
 
272: not their microbiomes but the microbiomes of patients with the respective diseases.  
 
278-279: Could the authors comment on the interesting fact that active CD is characterized with 
higher alpha -diversity than the inactive form? (despite the fact that inactive CD is clinically more 
resembling normal state). Also, the SCFA levels behave simi larly.  
 
279: Please revise: 16S rRNA sequencing does not allow to get data on amounts - just relative 
abundance.  
 
280-282: Consider a more specific and lighter statement instead of "aggressive":  the gut 
representatives of the phylum Bacteroidetes are most ly major commensal microbes.  
In the statement about butyrate, the fact itself is worth double -checking (Firmicutes are more 
capable in terms of butyrate, e.g. see https://mbio.asm.org/content/5/2/e00889 -14.abstract ), as 
well as the reference is not the mo st relevant.  
 
283: The reference [55] is not a review but a research study. Consider citing a more applicable 
meta -analysis/review of IBD -microbiome studies here.  
 
287: "Faecalibacterium, Oscillospira, and Feacalibacterium prausnitzii" - the list appear 
redundant - the first and the last items.  
 
283-338: As the Discussion section is too large, consider compacting this part containing mostly 
literature review with few direct links to the present findings.  
 
321: Consider choosing a consistent single term for each SCFA throughout the manuscript: e.g. 
butanoic acid and not butyrate, etc.  
 
348: Is there existing evidence of reverse: that host modulates microbiome via miRNA pool  
alterations?  
 
352: As in the case of microbes, it is the levels of miRNAs that were d ifferent between the 
groups, not the miRNAs themselves, isn't it?  
 
363: "inter -species" - is it about host -microbe, not microbe -microbe interaction?  
 
385: As far as the reviewer is concerned, correlations cannot have roles; it is just the reflection of 
the mechanisms with hidden causal links.  
 
 
 
The link to deposited metagenomic data is not valid (apparently, yet to be made public?).  
 
Figure 1: The figure is not very illustrative. Consider limiting to major OTUs, adding their 
taxonomic names, ordering the b arplots in a meaningful way. Besides, the colored frames 
(notation of "Phylum") are not visible, at least provided the image quality.  
 
Figure 2,3: consider keeping the same order of groups for boxplots (not to confuse the reader).  
 
Figure 5: Words are unreadable, together with low image quality and many layers deeming the 
whole picture incomprehensible. Should it be circular not oval also?  
 
Suppl Figure 1: consider arranging the plots in 3 x 2 grid.  
 
Suppl table 1: "baseMean - mean n ormalized count in all samples in experiment" - how was it 
calculated? Could it be dependent on read coverage? was rarefaction performed beforehand?  
There are multiple misprints "statistic", "p -vaue" in suppl. tables.  
 
Are the methods appropriate and well described?  
If not, plea se specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the dat a shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what as pects require further 
assessment in your comments to the editors.  
 
I am able to assess the statistics  
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of  competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1.  Have you in the past five years received reimbursements, f ees, funding, or salary 
from an organisation that may in any way gain or lose financial ly from the 
publication of this manuscript, either now or in the future?  
 
2.  Do you hold any stocks or shares in an organisation t hat may in any way gain or 
lose financially from the publication of this  manuscript, either now or in the 
future?  
 
3.  Do you hold or are you currently applying for any patents  relating to the content 
of the manuscript?  
 
4.  Have you received reimbursements, fees, funding, or salary from an organization 
that holds or has applied f or patents relating to the content of the manuscript?  
 
5.  Do you have any other financial competing interests?  
 
6.  Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have  no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests.  
 
I agree to the open peer review policy of the journal. I understan d that my name will be included 
on my report to the authors and, if the manuscript is accepted f or publication, my named report 
including any attachments I upload will be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be included in my named report can be in cluded as confidential comments 
to the editors, which will not be published.  
 
I agree to the open pee r review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
 
Title: In search for interplay between stool microRNAs, mi crobiota  and short chain fatty acids in 
Crohn’s disease. A preliminary study.  
 
Version: 0 Date: 07 Apr 2020  
 
Reviewer: Alexander V. Tyakht  
 
Reviewer's report:  
 
Grammar and language generally should be revised: some microbial taxa names are misspelled, 
punctuation signs missing, fragmented sentences, etc. Some are listed below.  
 
 
 
Last sentence in Abstract Background is fragmented.  
 
16s rRNA -> 16S rRNA (here and elsewhere)  
 
A. Results: It is not that the "taxa differed" rather that their relative abundance levels were 
different between the groups, isn't it? Consider rephrasing.  
 
Main text.  
 
41: 43 -45: Why is there an antithesis (" Although")? The two parts do not appear to contradict.  
 
49: Consider punctuation: "(IBDs) - Crohn's disease (CD) and ulcerative colitis (UC)".  
 
55: GaMmaproteobacteria  
 
57: FAecalibacterium prausnitziI  
 
59: 90% of the phylogenetic diversity? of the total m icrobial number? Please specify.  
Also, members of other phyla (e.g. Actinobacteria) can also produce SCFAs - consider not 
excluding them here.  
 
61: Dietary fibers are not the sole precursors of SCFA in the gut - at least another one being 
amino -acids resul ting from protein breakdown, perhaps to a lower extent: e.g. see 
https://sfamjournals.onlinelibrary.wiley.com/doi/pdf/10.1111/1462 -2920.13589 .  
 
67: Another linked reason worth mentioning is reduced fiber consumption in acute IBD patients. 
Consider includi ng, if possible, with reference.  
 
81: At which hospital were the patients enrolled and in which year? Is information about ethnic 
group available? (as genetics are an important factor contributing to CD risk)  
 
 
83: There is the first time bacterial mRNA ar e mentioned. If the bacterial gene expression has 
been conducted, it is an important expansion to the multi -omics study design, consider 
mentioning it in the Abstract, amending Introduction respectively.  
 
94: Please give more details on healthy controls as sessment. If it was only self -reported, without 
examination, did they at least complete Rome criteria or other well -being/gastrointestinal 
questionnaire?  
 
108: Consider listing the variable regions of the gene targeted by the kit.  
 
109: Perhaps, it is not that the bam files were unmapped in the sense of being unclassified - 
rather just "raw bam files"?  
Consider capitalizing fastq and fasta everywhere.  
 
125: If available, the data on Bristol stool scale should be included. At least, i f there were there 
any extreme values (diarrhea or very solid stool), I wonder how it affected the normalization of 
miRNA levels.  
 
166: It is suggested that the 16S rRNA microbiome survey term is used instead of metagenomics 
(the latter recommended to be r eserved for "shotgun" analysis only).  
 
167: Is it that the coverage was higher than targeted read count in all cases or there were >92000 
high-quality reads per sample?  
 
168: Consider changing: in more than 0.1% of reads -> at levels >0.1%  
 
173: I s it that Enterobacteriaceae was overrepresented in controls? Please reformulate.  
Did the method allow to distinguish Escherichia from Shigella based on 16S rRNA reads, as 
appears from the text? if not, consider changing to Escherichia/Shigella.  
 
189: What  does the genus name Prevotella_2 refer to? Perhaps, it is a notation from an older 
version of SILVA database - please check the latest release that has many of such issues 
resolved.  
 
201: Please explain the rationale behind this threshold. Can it be sugge sted from a 
distribution/histogram analysis as a separator between noise - and signal -level detection or 
elsewise?  
 
208: At least for these significant miRNA matches, please provide their length to orient the 
reader.  
 
Overall, could the authors provide more  interpretation related to the miRNAs associated with the 
disease? For example, is it possible to conduct some sort of enrichment analysis of them to look 
for functional explanation of these differences? Additional approach could be literature search if 
gut levels of them have been linked to this or other diseases.  
 
256-261: During all -to-all correlation analysis of multiple metabolites, miRNAs and microbial 
taxa, multiple comparison adjustment should have been performed. Please ensure that it has been 
done (does not appear from the Supplementary Table S6 at least). Considering significance, an 
adjusted p -value threshold should be examined, not correlation coefficient (apparently, threshold 
is > 0.6 here).  
 
269-270: "Opportunistic pathogens can also init iate IBD development through a process 
involving gut dysbiosis" - the reference does not support the statement: it just links acute enteric 
bacterial infection with chronic inflammatory disease, not IBD. Consider rephrasing or finding a 
more supporting ref erence.  
 
272: not their microbiomes but the microbiomes of patients with the respective diseases.  
 
278-279: Could the authors comment on the interesting fact that active CD is characterized with 
higher alpha -diversity than the inactive form? (despite the fact that inactive CD is clinically more 
resembling normal state). Also, the SCFA levels behave simi larly.  
 
279: Please revise: 16S rRNA sequencing does not allow to get data on amounts - just relative 
abundance.  
 
280-282: Consider a more specific and lighter statement instead of "aggressive":  the gut 
representatives of the phylum Bacteroidetes are most ly major commensal microbes.  
In the statement about butyrate, the fact itself is worth double -checking (Firmicutes are more 
capable in terms of butyrate, e.g. see https://mbio.asm.org/content/5/2/e00889 -14.abstract ), as 
well as the reference is not the mo st relevant.  
 
283: The reference [55] is not a review but a research study. Consider citing a more applicable 
meta -analysis/review of IBD -microbiome studies here.  
 
287: "Faecalibacterium, Oscillospira, and Feacalibacterium prausnitzii" - the list appear 
redundant - the first and the last items.  
 
283-338: As the Discussion section is too large, consider compacting this part containing mostly 
literature review with few direct links to the present findings.  
 
321: Consider choosing a consistent single term for each SCFA throughout the manuscript: e.g. 
butanoic acid and not butyrate, etc.  
 
348: Is there existing evidence of reverse: that host modulates microbiome via miRNA pool  
alterations?  
 
352: As in the case of microbes, it is the levels of miRNAs that were d ifferent between the 
groups, not the miRNAs themselves, isn't it?  
 
363: "inter -species" - is it about host -microbe, not microbe -microbe interaction?  
 
385: As far as the reviewer is concerned, correlations cannot have roles; it is just the reflection of 
the mechanisms with hidden causal links.  
 
 
 
The link to deposited metagenomic data is not valid (apparently, yet to be made public?).  
 
Figure 1: The figure is not very illustrative. Consider limiting to major OTUs, adding their 
taxonomic names, ordering the b arplots in a meaningful way. Besides, the colored frames 
(notation of "Phylum") are not visible, at least provided the image quality.  
 
Figure 2,3: consider keeping the same order of groups for boxplots (not to confuse the reader).  
 
Figure 5: Words are unreadable, together with low image quality and many layers deeming the 
whole picture incomprehensible. Should it be circular not oval also?  
 
Suppl Figure 1: consider arranging the plots in 3 x 2 grid.  
 
Suppl table 1: "baseMean - mean n ormalized count in all samples in experiment" - how was it 
calculated? Could it be dependent on read coverage? was rarefaction performed beforehand?  
There are multiple misprints "statistic", "p -vaue" in suppl. tables.  
 
Are the methods appropriate and well described?  
If not, plea se specify what is required in your comments to the authors.  
 
Yes 
 
Does the work include the necessary controls?  
If not, please specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the dat a shown?  
If not, please explain in your comments to the authors.  
 
Yes 
 
Are you able to assess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please specify what as pects require further 
assessment in your comments to the editors.  
 
I am able to assess the statistics  
Quality of written English  
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of  competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1.  Have you in the past five years received reimbursements, f ees, funding, or salary 
from an organisation that may in any way gain or lose financial ly from the 
publication of this manuscript, either now or in the future?  
 
2.  Do you hold any stocks or shares in an organisation t hat may in any way gain or 
lose financially from the publication of this  manuscript, either now or in the 
future?  
 
3.  Do you hold or are you currently applying for any patents  relating to the content 
of the manuscript?  
 
4.  Have you received reimbursements, fees, funding, or salary from an organization 
that holds or has applied f or patents relating to the content of the manuscript?  
 
5.  Do you have any other financial competing interests?  
 
6.  Do you have any non -financial competing interests in relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have  no competing interests' 
below. If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests.  
 
I agree to the open peer review policy of the journal. I understan d that my name will be included 
on my report to the authors and, if the manuscript is accepted f or publication, my named report 
including any attachments I upload will be posted on the website along with the authors' 
responses. I agree for my report to be made available under an Open Access Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments 
which I do not wish to be included in my named report can be in cluded as confidential comments 
to the editors, which will not be published.  
 
I agree to the open pee r review policy of the journal  
assistant