Review for 10.1186/1471-230X-10-134 (2010)

Reviewer's report
Title:Ulcerative colitis and irritable bowel patients exhibit distinct abnormalities of
the gut microbiota
Version: 1Date:1 March 2010
Reviewer: Grietje Holtrop
Reviewer's report:
STATISTICAL REVIEW
The alignment and scaling of DGGE gels has been done in a thorough manner.
Unfortunately, subsequent statistical analyses are poorly explained and not
well-justified, and the methodology used is inappropriate in places (such as
t-tests instead of analysis of variance). More details are given below.
COMPULSORY REVISION
C1. How was the replication mentioned in comment E1 taken into account in the
statistical analyses? Please clarify.
C2. Second page of Results, line 3 onwards and elsewhere: there are three
treatment groups and these data should be analysed by Analysis of Variance
(ANOVA). If the effect of Treatment is significant, post-hoc t-tests (i.e. based on
the mean residual sum of squares and residual degrees of freedom from the
ANOVA output) can be performed to investigate which of the treatments differ.
Replicate samples obtained from the same subject should be taken into account
appropriately, this to avoid overly significant findings.
C3. Were assumptions of normality and equality of variance (with the latter begin
more important than the former) across the treatment groups investigated? Were
transformations or alternative analyses considered when these two assumptions
were not met?
C4. Second page of Results, 2nd paragraph 2nd line: how was P < 10E-6
obtained?
C5. (see also comment E4) If PLS is already based on class membership, then
what additional information is provided by the CVA analysis?
C6. CVA: how were the data ‘condensed’ before implementing CVA (as there are
many more RF variables than samples)? Was this done by principal component
analysis? How many principal components were used in the subsequent CVA?
Please explain.
C7. Fourth page of Materials & Methods, line 6-7: why were the DGGE bands
treated as absence/presence data for calculating similarity indices, whilst for the
Shannon diversity index the band intensity was taken into account? If band
intensity is regarded reliable then why was the similarity index calculated from
absence/presence?
C8. Why were bands to be excised selected based on the first PLS component
only? Why was this not based on the Euclidean length of each RF in the first four
PLS dimensions, say (based on classification success rates in Fig 3a)? If CVA
gives the optimal 2-dimensional separation of the three treatment groups, then
why was band-selection not based on maximum peaks from the first CV axis
(back-transformed to the original RF) instead?
C9. Why were four bands selected for sequencing? Fig 4a seems to suggest that
there are a further three peaks (0.221, 0.644, 0.746) that may be influential in
segregation of the treatment groups. Were the four top-ranking bands always the
same in the cross-validation exercise?
C10. Third page of Results section, 2nd paragraph (similarity). How were the
similarity data analysed statistically? How were Pvalues obtained? It should be
noted that we cannot perform t-tests or ANOVA on similarities, as there will be
many more similarities than data points (for example, 4 data points will give 6
similarities, but t-tests/anova can not be used as the similarities are not
independent from one another).
MINOR ESSENTIAL REVISION
E1. Please specify how many faecal samples per subject were analysed. At the
start of Materials & Methods we have 13+11+22=46 subjects, whilst the data
matrix based on DGGE mentions 96 lanes.The number of data points in Figure
3b also suggests that there were multiple samples per volunteer. Please also
specify whether the multiple DGGE lanes were replicates from the same faecal
sample or consisted of faecal samples collected at different days but from the
same volunteer.
E2. Please explain the statistical methodology in layman’s terms, as many
readers with a microbiology background will not be familiar with metabolomics
techniques such as PLS.
E3. Second page of Results, line 6 onwards: why is the median reported here
instead of the mean?
E4. Third page of Materials & Methods, bottom line: Please mention what was
used as the Y-variable. Was this the so-called ‘class membership’?
E5. Is the information presented in Figure 3a used at all? See also comment C8.
E6. For how many samples and/or volunteers were the four selected bands
sequenced? Was this done for all samples or for a subset of samples? If done for
a subset of samples, how were the samples selected? Please clarify
E7. Materials & Methods, Subjects and sample collection. Line 4 states ‘age
18-60’ whilst line 6 gives ranges up to 64 years. Please make this consistent.
E8. Fig 6: what do the error bars represent?
E9. Fig 3a: the permutation test results are not well-explained. What do the mean
+- 2 SD represent? Why are the means always approximately 37%, what does
this mean?
E10. What does the +- refer to in the text (e.g. bottom line of first page of Results
and various other places)?
DISCRETIONARY REVISION
None
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: Yes, and I have assessed the statistics in my report.
Declaration of competing interests:
I declare that I have no competing interests