Reviewer ’s report  
 
Title: Evaluating gut microbiota profiles from archived fecal samples  
 
Version: 0 Date: 19 Jul 2018  
 
Reviewer: Alexandra Zhernakova  
 
Reviewer's report:  
 
The paper by Rounge et al describes the i mportant topic of stability and variations in microbiome 
results from archived, iFOBT and fresh fecal samples, as well as the effect of storage and 
possible contamination.  
 
The paper addresses the important issue, relevant to the methodology of microbiome  studies, and 
the results are of interest to the broad audience. In general, the introduction and discussion parts 
are well written, however, the results part is less clear; I suggest several major changes.  
 
The paper represents a number of pairwise compar isons of samples (archived, iFOBT, fresh, 
storage time, etc), however different comparisons are done using different workflow and the data 
presentation is not always the same. I suggest making the results part more systematic, by 
describing the differences  in alpha diversity, beta diversity and OTU abundances across all 
comparisons.  
 
The paper will benefit from PCoA plot where all samples will be indicated by different color on 
one plot.  
 
Moreover, it will be useful to make one table with the list of OTU abundances in every sample 
type. Although the difference in OTU abundances across comparison can be not significant due 
to the low number of samples, it will be useful to present all the da ta in a joint table; This table 
can be used by researchers as a reference of which OTUs are dependent on the storage method.  
 
 
 
Minor comments:  
 
"A microbiome biomarker based on 16S sequencing combined with FOBT screening was shown 
to increase CRC detection  compared to iFOBT/FOBT screening." - please add the reference to 
this sentence.  
 
"In total, 4.2 million 16S rDNA contigs were assembled from the sequencing data. The fresh 
samples were sequenced deeper, thus producing more contigs and OTUs (Figure 1A, Ad ditional 
file 3 and 4)." - please explain clearly in the results section why the fresh samples were 
sequenced deeper compared to (which samples?)  
 
"Archived samples produced less OTUs than fresh and iFOBT samples" - give reference to table 
1. 
 
"The mean nu mbers of OTUs with a count >4 differed for the sample series" - this sentence is 
unclear.  
 
"passed a minimum of 200 OTUs at first attempt" - what do you mean with "at first attempt"?  
 
Table 1 is not easy to read, I suggest the following editing: 1. Put mea n/min/max as separate 
columns, 2. Add % to the last two columns.  
 
Comparison of samples stored at room temperature: The authors report only the comparison of 
alpha diversity; what about Bray -Curtis plots and comparison of specific taxa? This is important 
to add.  
 
I agree with conclusions, however, it is important to clearly state that storage and sample method 
has a substantial effect on the identified microbiome composition and diversity, therefore in the 
case-control design, both cases and controls shou ld be processed in the same way.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
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 data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to as sess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please speci fy what aspects 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:  
 
Acceptable  
 
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 recei ved reimbursements, fees, funding, or salary from an organization that 
holds or has applied for 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 r eview 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 c omments 
which I do not wish to be included in my named report can be included a s confidential comments 
to the editor s, which will not be published.  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
 
Title: Evaluating gut microbiota profiles from archived fecal samples  
 
Version: 0 Date: 19 Jul 2018  
 
Reviewer: Alexandra Zhernakova  
 
Reviewer's report:  
 
The paper by Rounge et al describes the i mportant topic of stability and variations in microbiome 
results from archived, iFOBT and fresh fecal samples, as well as the effect of storage and 
possible contamination.  
 
The paper addresses the important issue, relevant to the methodology of microbiome  studies, and 
the results are of interest to the broad audience. In general, the introduction and discussion parts 
are well written, however, the results part is less clear; I suggest several major changes.  
 
The paper represents a number of pairwise compar isons of samples (archived, iFOBT, fresh, 
storage time, etc), however different comparisons are done using different workflow and the data 
presentation is not always the same. I suggest making the results part more systematic, by 
describing the differences  in alpha diversity, beta diversity and OTU abundances across all 
comparisons.  
 
The paper will benefit from PCoA plot where all samples will be indicated by different color on 
one plot.  
 
Moreover, it will be useful to make one table with the list of OTU abundances in every sample 
type. Although the difference in OTU abundances across comparison can be not significant due 
to the low number of samples, it will be useful to present all the da ta in a joint table; This table 
can be used by researchers as a reference of which OTUs are dependent on the storage method.  
 
 
 
Minor comments:  
 
"A microbiome biomarker based on 16S sequencing combined with FOBT screening was shown 
to increase CRC detection  compared to iFOBT/FOBT screening." - please add the reference to 
this sentence.  
 
"In total, 4.2 million 16S rDNA contigs were assembled from the sequencing data. The fresh 
samples were sequenced deeper, thus producing more contigs and OTUs (Figure 1A, Ad ditional 
file 3 and 4)." - please explain clearly in the results section why the fresh samples were 
sequenced deeper compared to (which samples?)  
 
"Archived samples produced less OTUs than fresh and iFOBT samples" - give reference to table 
1. 
 
"The mean nu mbers of OTUs with a count >4 differed for the sample series" - this sentence is 
unclear.  
 
"passed a minimum of 200 OTUs at first attempt" - what do you mean with "at first attempt"?  
 
Table 1 is not easy to read, I suggest the following editing: 1. Put mea n/min/max as separate 
columns, 2. Add % to the last two columns.  
 
Comparison of samples stored at room temperature: The authors report only the comparison of 
alpha diversity; what about Bray -Curtis plots and comparison of specific taxa? This is important 
to add.  
 
I agree with conclusions, however, it is important to clearly state that storage and sample method 
has a substantial effect on the identified microbiome composition and diversity, therefore in the 
case-control design, both cases and controls shou ld be processed in the same way.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
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 data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to as sess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please speci fy what aspects 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:  
 
Acceptable  
 
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 recei ved reimbursements, fees, funding, or salary from an organization that 
holds or has applied for 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 r eview 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 c omments 
which I do not wish to be included in my named report can be included a s confidential comments 
to the editor s, which will not be published.  
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
 
Title: Evaluating gut microbiota profiles from archived fecal samples  
 
Version: 0 Date: 19 Jul 2018  
 
Reviewer: Alexandra Zhernakova  
 
Reviewer's report:  
 
The paper by Rounge et al describes the i mportant topic of stability and variations in microbiome 
results from archived, iFOBT and fresh fecal samples, as well as the effect of storage and 
possible contamination.  
 
The paper addresses the important issue, relevant to the methodology of microbiome  studies, and 
the results are of interest to the broad audience. In general, the introduction and discussion parts 
are well written, however, the results part is less clear; I suggest several major changes.  
 
The paper represents a number of pairwise compar isons of samples (archived, iFOBT, fresh, 
storage time, etc), however different comparisons are done using different workflow and the data 
presentation is not always the same. I suggest making the results part more systematic, by 
describing the differences  in alpha diversity, beta diversity and OTU abundances across all 
comparisons.  
 
The paper will benefit from PCoA plot where all samples will be indicated by different color on 
one plot.  
 
Moreover, it will be useful to make one table with the list of OTU abundances in every sample 
type. Although the difference in OTU abundances across comparison can be not significant due 
to the low number of samples, it will be useful to present all the da ta in a joint table; This table 
can be used by researchers as a reference of which OTUs are dependent on the storage method.  
 
 
 
Minor comments:  
 
"A microbiome biomarker based on 16S sequencing combined with FOBT screening was shown 
to increase CRC detection  compared to iFOBT/FOBT screening." - please add the reference to 
this sentence.  
 
"In total, 4.2 million 16S rDNA contigs were assembled from the sequencing data. The fresh 
samples were sequenced deeper, thus producing more contigs and OTUs (Figure 1A, Ad ditional 
file 3 and 4)." - please explain clearly in the results section why the fresh samples were 
sequenced deeper compared to (which samples?)  
 
"Archived samples produced less OTUs than fresh and iFOBT samples" - give reference to table 
1. 
 
"The mean nu mbers of OTUs with a count >4 differed for the sample series" - this sentence is 
unclear.  
 
"passed a minimum of 200 OTUs at first attempt" - what do you mean with "at first attempt"?  
 
Table 1 is not easy to read, I suggest the following editing: 1. Put mea n/min/max as separate 
columns, 2. Add % to the last two columns.  
 
Comparison of samples stored at room temperature: The authors report only the comparison of 
alpha diversity; what about Bray -Curtis plots and comparison of specific taxa? This is important 
to add.  
 
I agree with conclusions, however, it is important to clearly state that storage and sample method 
has a substantial effect on the identified microbiome composition and diversity, therefore in the 
case-control design, both cases and controls shou ld be processed in the same way.  
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
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 data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to as sess any statistics in the manus cript or would you recommend an 
additional statistical review?  
If an additional statistical review is recommended, please speci fy what aspects 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:  
 
Acceptable  
 
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 recei ved reimbursements, fees, funding, or salary from an organization that 
holds or has applied for 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 r eview 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 c omments 
which I do not wish to be included in my named report can be included a s confidential comments 
to the editor s, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant