Review for 10.1186/1471-230X-9-95 (2009)

Reviewer's report
Title:Microbial community analysis reveals high level phylogenetic alterations in
the overall gastrointestinal microbiota of diarrhoea-predominant irritable bowel
syndrome sufferers
Version: 3Date:15 October 2009
Reviewer: Petra Louis
Reviewer's report:
Krogius-Kurikka et al have improved their manuscript on clone library analysis of
IBS patients vs healthy individuals and the limitations of the study are addressed
to a degree, however, further changes are necessary before the manuscript is
acceptable for publication.
Major compulsory revisions:
1. Point 1, Comparison of samples pooled from different numbers of volunteers: I
principally agree with the changed emphasis on the presentation of the data,
however, only showing the diversity indices at 98% phylotype level (i.e. at the
level that shows up the higher diversity of predominantly low-abundance OTUs
due to higher sample number in the HC group) means that only the diversity data
that are most questionable are shown. I would therefore suggest to include
additional wording to the main text (p. 14, results, library characteristics, line 9; or
in the discussion) mentioning that the libraries differed less at lower OTU cut-offs
and that the likely cause (or at least contributor) to the differences seen is the
difference in numbers of individuals per library.
2. Point 2, Skew due to unproportioned fractionation: I cannot fully follow the
comparison of proportioned to unproportioned comparison of a fractionated
library. The authors state data from Krogius-Kurikka et al, however, Table 2
compared the fractioned library to a smaller unfractioned library, which is a
different comparison to the one I am thinking about. As I don’t have the data I
cannot judge how the values given for the comparison in the authors response
were derived, however, I find it difficult to understand how DNA abundance
differences of a factor of up to approx. 7-fold between fractions could have such
little effect.
As for the author’s 2. comment within point 2, I was not suggesting that the
relative amount of DNA in each fraction reflects the number of subjects, but was
referring to the difference in relative abundance plots in the 2 libraries (additional
file 1) leading to further skew if each fraction is treated equally.
In any case, it has to be made clear in the discussion (p. 17, 1. paragraph) that
the equal treatment of DNA fractions of different abundance may lead to some
skew of the relative abundance of bacterial groups/OTUs. The authors
themselves have indicated this in their previous paper.
3. Point 7, comparison to real-time PCR data: Differences between IBS-D and
HC at phylum and family level are reported here based on clone library analysis.
Therefore, the discussion should include a comparison to previous real-time PCR
data on higher-level groups (e.g. Malinen et al, 2005,
Bacteroides-Prevotella-Porphyromonas, C. coccoides group).
4. Point 9: I am still not clear about the numbers in Tab. 1 and Fig. 4. Fig. 4
allows the calculation of OTUs represented by IBS-D and HC, respectively, by
adding the unique and shared OTUs for each library. This leads to 314 OTUs for
IBS-D and 440 OTUs for HC (with 176 of those OTUs shared). Should these
numbers not be in agreement with the individual libraries in Table 1? The only
reason I can think of to explain the discrepancy between individual and joint
analysis of the libraries is that the 2 analyses (each one individually or both
combined) could have lead to some sequences falling into different OTU groups,
thus differing in the total number of OTUs? If this is the case, would it not be
more meaningful to analyse only the combined library, as these differences must
be due to a technical issue (e.g. differences in the alignments between the
individual libraries due to presence/absence of certain sequences)?
Minor essential revisions:
1. In the abstract (methods, 2. sentence), please include number of individuals
for healthy control library.
2. Abstract, conclusions: I don’t understand the meaning of the last sentence.
The characteristics were more distinct in comparison to what? The distinctions to
the HC library are already mentioned in the previous sentence.
3. Point 4, real-time PCR: Please provide further information in Methods (any in
silico methods used for primer design, clone used as negative control) or refer to
Rinttila et al 2004 where appropriate.
4. Point 10: Please include a brief statement in the results or discussion that the
moisture content did not affect the results.
5. Discussion p. 17, line 4 of 2. paragraph: please delete ‘thus’, as it does not
make sense in the modified text.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests