Reviewer ’s report  
 
Title: Evaluating gut microbiota profiles from archived fecal samples  
 
Version: 0 Date: 17 Jul 2018  
 
Reviewer: Robert Kraaij  
 
Reviewer's report:  
 
The manuscript "Evaluating gut microbiota profiles from archived fecal samples" by Rounge et 
al. is mostly well written and addresses an important issue of using archived samples from CRC 
screening trials to perform large -scale, longitudinal studies on gut  microbiota. This paper 
evaluates the feasibility of such materials to be used in such studies.  
 
It is already known that iFOBT samples can be used to generate microbiome composition data, 
as described in the Discussion section (line 288). But this analysi s on the 50 iFOBT samples 
would add to our knowledge if it was properly analyzed. The effects of long term storage up to 
16 years at -30C has, also to my opinion, not been published before but need some additional and 
better analyses. The effects of 48h at  room temperature are already known.  
 
I have some major issues with this manuscript, although it is important enough to be 
communicated to the scientific community. The assessment of quality of stool samples that have 
been at -30C for up to 16 years is an  important issue. Furthermore, assessment of the quality of 
stool samples that have been collected through iFOBT (small stool sample in large volume of 
buffer) is important since there are large numbers of such samples world -wide. Why the 
assessment of the  effects of storage at room temperature for 48h is included has not been 
described well in the manuscript and is not clear to me.  
 
 
Major comments:  
 
1) In the Background, I would like to see a better description of the different sample groups that 
are avai lable, what issues are being addressed in the paper, and why these issues are important.  
 
Minor comment on the description of the groups:  
- line 120: what happened to the samples after arrival in the lab.  
 
2) Paragraphs addressing the contamination during iFOBT analysis are clear. (No major 
comment, I just listed this one to be complete)  
 
3) Paragraphs addressing evaporation, sublimation and condensation are not clear. First, it is not 
directly clear to me what the differences are between evaporation and s ublimation. Second, why 
do you think this is a problem: are you expecting to lose half of the volume? Third, what are you 
expecting to observe in this experiment, as the results show an increase in weight, likely due to 
condensation? Since the problem has not been defined well and the results are opposite of what I 
was expecting, I would like to see better explanations of this experiment and its results.  
 
4) Paragraphs addressing OTU table generation are not clear. Has the OTU table been 
normalized in any of the analyses? This is not clear to me and I have the feeling that samples, 
that have been sequenced at different depths and thus have different numbers of OTUs just as a 
consequences of sequencing depth, have been compared. In line 219, the authors ment ion the 
OTU cut point but it is not clear what it is, how it was determined and what has been done with 
this cut point. To better understand the subsequent analyses it is important to know what "has 
been done" to the OTU table that has been used. Maybe the se are standard procedures in the 
Mother pipeline, but then I would still like to obtain some explanation on the methods.  
 
Some minor comments on this:  
- line 179: specify the version of the Silva database  
- line 180: is this closed reference or open refe rence OTU calling?  
 
5) Paragraphs addressing alpha -diversity and composition between the groups are not clear. 
Based on the presented data it is clear that the Archived samples perform less well in the PCR 
and during sequencing (number of reads per sample). The iFOBT group is  intermediate and the 
Fresh samples performed best. But despite this technical issue, the main question is whether the 
compositions are significantly different.  
 
5a) In Fig. 1 the effects on number of species (A) and alpha -diversity (B and C) are shown. A re 
Figs. 1 A, B and C based on normalized data?  
 
5b) Fig. 1D, E and F address the important question whether the profiles differ significantly 
between the three groups based on the pair -wise beta -diversities. First, this should not be done 
while including sample pairs in one group (see line 314). Second, this should be performed by 
PERMANOVA like the "adonis" function in Vegan which will give explained variance as well 
as statistics.  
 
Minor comment on the preservation:  
-  does sodium azide (line 318) prese rves the sample? Can you comment on that in the 
Discussion section?  
 
6) Experiments addressing the effect of 2 days at room temperature are not well analyzed. In the 
Results section (lines 265 -273), first alpha -diversity is addressed, then beta/composition  and then 
alpha again. A graph showing the effect on alpha (like additional file 2) would be more 
informative, although not significant, than Fig. 3. Second, for the composition analysis using 
clustering, stating that "inter -person variability is higher th an intra -person variability" is not the 
proper way to analyze these results as inter -person variability is high for the gut microbiome. I 
am not sure how to analyze this, but calculating the Bray -Curtis dissimilarities between the 
sample pairs and compare those to technical replicates or data from the literature would be a 
start. Maybe the PERMANOVA will work too.  
 
 
 
 
 
 
 
 
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 recommend additiona l statistical review  
 
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: 17 Jul 2018  
 
Reviewer: Robert Kraaij  
 
Reviewer's report:  
 
The manuscript "Evaluating gut microbiota profiles from archived fecal samples" by Rounge et 
al. is mostly well written and addresses an important issue of using archived samples from CRC 
screening trials to perform large -scale, longitudinal studies on gut  microbiota. This paper 
evaluates the feasibility of such materials to be used in such studies.  
 
It is already known that iFOBT samples can be used to generate microbiome composition data, 
as described in the Discussion section (line 288). But this analysi s on the 50 iFOBT samples 
would add to our knowledge if it was properly analyzed. The effects of long term storage up to 
16 years at -30C has, also to my opinion, not been published before but need some additional and 
better analyses. The effects of 48h at  room temperature are already known.  
 
I have some major issues with this manuscript, although it is important enough to be 
communicated to the scientific community. The assessment of quality of stool samples that have 
been at -30C for up to 16 years is an  important issue. Furthermore, assessment of the quality of 
stool samples that have been collected through iFOBT (small stool sample in large volume of 
buffer) is important since there are large numbers of such samples world -wide. Why the 
assessment of the  effects of storage at room temperature for 48h is included has not been 
described well in the manuscript and is not clear to me.  
 
 
Major comments:  
 
1) In the Background, I would like to see a better description of the different sample groups that 
are avai lable, what issues are being addressed in the paper, and why these issues are important.  
 
Minor comment on the description of the groups:  
- line 120: what happened to the samples after arrival in the lab.  
 
2) Paragraphs addressing the contamination during iFOBT analysis are clear. (No major 
comment, I just listed this one to be complete)  
 
3) Paragraphs addressing evaporation, sublimation and condensation are not clear. First, it is not 
directly clear to me what the differences are between evaporation and s ublimation. Second, why 
do you think this is a problem: are you expecting to lose half of the volume? Third, what are you 
expecting to observe in this experiment, as the results show an increase in weight, likely due to 
condensation? Since the problem has not been defined well and the results are opposite of what I 
was expecting, I would like to see better explanations of this experiment and its results.  
 
4) Paragraphs addressing OTU table generation are not clear. Has the OTU table been 
normalized in any of the analyses? This is not clear to me and I have the feeling that samples, 
that have been sequenced at different depths and thus have different numbers of OTUs just as a 
consequences of sequencing depth, have been compared. In line 219, the authors ment ion the 
OTU cut point but it is not clear what it is, how it was determined and what has been done with 
this cut point. To better understand the subsequent analyses it is important to know what "has 
been done" to the OTU table that has been used. Maybe the se are standard procedures in the 
Mother pipeline, but then I would still like to obtain some explanation on the methods.  
 
Some minor comments on this:  
- line 179: specify the version of the Silva database  
- line 180: is this closed reference or open refe rence OTU calling?  
 
5) Paragraphs addressing alpha -diversity and composition between the groups are not clear. 
Based on the presented data it is clear that the Archived samples perform less well in the PCR 
and during sequencing (number of reads per sample). The iFOBT group is  intermediate and the 
Fresh samples performed best. But despite this technical issue, the main question is whether the 
compositions are significantly different.  
 
5a) In Fig. 1 the effects on number of species (A) and alpha -diversity (B and C) are shown. A re 
Figs. 1 A, B and C based on normalized data?  
 
5b) Fig. 1D, E and F address the important question whether the profiles differ significantly 
between the three groups based on the pair -wise beta -diversities. First, this should not be done 
while including sample pairs in one group (see line 314). Second, this should be performed by 
PERMANOVA like the "adonis" function in Vegan which will give explained variance as well 
as statistics.  
 
Minor comment on the preservation:  
-  does sodium azide (line 318) prese rves the sample? Can you comment on that in the 
Discussion section?  
 
6) Experiments addressing the effect of 2 days at room temperature are not well analyzed. In the 
Results section (lines 265 -273), first alpha -diversity is addressed, then beta/composition  and then 
alpha again. A graph showing the effect on alpha (like additional file 2) would be more 
informative, although not significant, than Fig. 3. Second, for the composition analysis using 
clustering, stating that "inter -person variability is higher th an intra -person variability" is not the 
proper way to analyze these results as inter -person variability is high for the gut microbiome. I 
am not sure how to analyze this, but calculating the Bray -Curtis dissimilarities between the 
sample pairs and compare those to technical replicates or data from the literature would be a 
start. Maybe the PERMANOVA will work too.  
 
 
 
 
 
 
 
 
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 recommend additiona l statistical review  
 
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: 17 Jul 2018  
 
Reviewer: Robert Kraaij  
 
Reviewer's report:  
 
The manuscript "Evaluating gut microbiota profiles from archived fecal samples" by Rounge et 
al. is mostly well written and addresses an important issue of using archived samples from CRC 
screening trials to perform large -scale, longitudinal studies on gut  microbiota. This paper 
evaluates the feasibility of such materials to be used in such studies.  
 
It is already known that iFOBT samples can be used to generate microbiome composition data, 
as described in the Discussion section (line 288). But this analysi s on the 50 iFOBT samples 
would add to our knowledge if it was properly analyzed. The effects of long term storage up to 
16 years at -30C has, also to my opinion, not been published before but need some additional and 
better analyses. The effects of 48h at  room temperature are already known.  
 
I have some major issues with this manuscript, although it is important enough to be 
communicated to the scientific community. The assessment of quality of stool samples that have 
been at -30C for up to 16 years is an  important issue. Furthermore, assessment of the quality of 
stool samples that have been collected through iFOBT (small stool sample in large volume of 
buffer) is important since there are large numbers of such samples world -wide. Why the 
assessment of the  effects of storage at room temperature for 48h is included has not been 
described well in the manuscript and is not clear to me.  
 
 
Major comments:  
 
1) In the Background, I would like to see a better description of the different sample groups that 
are avai lable, what issues are being addressed in the paper, and why these issues are important.  
 
Minor comment on the description of the groups:  
- line 120: what happened to the samples after arrival in the lab.  
 
2) Paragraphs addressing the contamination during iFOBT analysis are clear. (No major 
comment, I just listed this one to be complete)  
 
3) Paragraphs addressing evaporation, sublimation and condensation are not clear. First, it is not 
directly clear to me what the differences are between evaporation and s ublimation. Second, why 
do you think this is a problem: are you expecting to lose half of the volume? Third, what are you 
expecting to observe in this experiment, as the results show an increase in weight, likely due to 
condensation? Since the problem has not been defined well and the results are opposite of what I 
was expecting, I would like to see better explanations of this experiment and its results.  
 
4) Paragraphs addressing OTU table generation are not clear. Has the OTU table been 
normalized in any of the analyses? This is not clear to me and I have the feeling that samples, 
that have been sequenced at different depths and thus have different numbers of OTUs just as a 
consequences of sequencing depth, have been compared. In line 219, the authors ment ion the 
OTU cut point but it is not clear what it is, how it was determined and what has been done with 
this cut point. To better understand the subsequent analyses it is important to know what "has 
been done" to the OTU table that has been used. Maybe the se are standard procedures in the 
Mother pipeline, but then I would still like to obtain some explanation on the methods.  
 
Some minor comments on this:  
- line 179: specify the version of the Silva database  
- line 180: is this closed reference or open refe rence OTU calling?  
 
5) Paragraphs addressing alpha -diversity and composition between the groups are not clear. 
Based on the presented data it is clear that the Archived samples perform less well in the PCR 
and during sequencing (number of reads per sample). The iFOBT group is  intermediate and the 
Fresh samples performed best. But despite this technical issue, the main question is whether the 
compositions are significantly different.  
 
5a) In Fig. 1 the effects on number of species (A) and alpha -diversity (B and C) are shown. A re 
Figs. 1 A, B and C based on normalized data?  
 
5b) Fig. 1D, E and F address the important question whether the profiles differ significantly 
between the three groups based on the pair -wise beta -diversities. First, this should not be done 
while including sample pairs in one group (see line 314). Second, this should be performed by 
PERMANOVA like the "adonis" function in Vegan which will give explained variance as well 
as statistics.  
 
Minor comment on the preservation:  
-  does sodium azide (line 318) prese rves the sample? Can you comment on that in the 
Discussion section?  
 
6) Experiments addressing the effect of 2 days at room temperature are not well analyzed. In the 
Results section (lines 265 -273), first alpha -diversity is addressed, then beta/composition  and then 
alpha again. A graph showing the effect on alpha (like additional file 2) would be more 
informative, although not significant, than Fig. 3. Second, for the composition analysis using 
clustering, stating that "inter -person variability is higher th an intra -person variability" is not the 
proper way to analyze these results as inter -person variability is high for the gut microbiome. I 
am not sure how to analyze this, but calculating the Bray -Curtis dissimilarities between the 
sample pairs and compare those to technical replicates or data from the literature would be a 
start. Maybe the PERMANOVA will work too.  
 
 
 
 
 
 
 
 
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 recommend additiona l statistical review  
 
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