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Inflammation-associated enterotypes, host genotype, cage and inter-individual effects drive gut microbiota variation in common laboratory mice

Falk Hildebrand12, Thi Loan Anh Nguyen1234, Brigitta Brinkman56, Roberto Garcia Yunta12, Benedicte Cauwe34, Peter Vandenabeele56, Adrian Liston34 and Jeroen Raes12*

Author Affiliations

1 Department of Structural Biology, VIB, Pleinlaan 2, 1050 Brussels, Belgium

2 Department of Bioscience Engineering, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium

3 Autoimmune Genetics Laboratory, VIB, Herestraat 49, 3000 Leuven, Belgium

4 Katholieke Universiteit Leuven, Herestraat 49, 3000 Leuven, Belgium

5 Department for Molecular Biomedical Research, VIB, Technologiepark Zwijnaarde 927, 9052 Ghent, Belgium

6 Department for Molecular Biomedical Research, GhentUniversity, Technologiepark Zwijnaarde 927, 9052 Ghent, Belgium

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Genome Biology 2013, 14:R4  doi:10.1186/gb-2013-14-1-r4

Published: 24 January 2013

Additional files

Additional file 1:

Figure S1 - overview of gut microbiome composition of investigated samples at the phylum level. Mouse strains are abreviated by the first letters and correspond in color to Figure 1a: N, NOD; F, FVB; BA, Balbc; S, Swiss; B, B6.

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Additional file 2:

Figure S2 - density plotting of samples on NMDS revealed two enterotypes at the phylum level. The same result, that is, two optimal clusters, was observed when using three different distance matrices: (a) genus level Bray-Curtis, (b) genus level Jensen-Shannon and (c) OTU level weighted Unifrac.

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Additional file 3:

Table S1 - distribution of enterotypes among genotypes and cages. Distribution of enterotypes among (a) genotypes and (b) cages. Enterotype 1 and 2 are labeled as ET1 and ET2, respectively.

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Additional file 4:

Table S2 - optimal clustering numbers of the total dataset. (a) The optimal number of clusters obtained by Silhouette index/Calinski-Harabasz (CH) score. (b) The actual observed CH score. (c) The observed maximum Silhouette index. This is repeated at five taxonomic levels using four different distance methods. Note that Unifrac distance can only be measured at the OTU level.

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Additional file 5:

Table S3 - comparison of optimal cluster number under differing clustering methods as well as optimal cluster number scores. All data are calculated at the genus level, using Jensen-Shannon distance. Abbrevations: CH, Calinski-Harabasz pseudo F-statistic; SIL, Silhouette internal cluster optimality criterion; BHG, Baker and Hubert Gamma; DB, Davies-Bouldin's index.

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Additional file 6:

Table S4 - 10% of the taxonomy was either resampled or the samples were jackniffed to 54 samples. This was repeated 500 times under 5 clustering conditions using pam clustering and the taxonomic level as indicated. The optimal cluster number in these 500 resamplings is shown in the tables. Abbrevations: CH, Calinski-Harabasz pseudo F-statistic; SIL, Silhouette internal cluster.

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Additional file 7:

Table S5 - taxa that showed significant differences between enterotype 1 (ET1) and enterotype 2 (ET2). Cut-off values (P < 0.05 and q < 0.1) were applied. The OTUs were identified at the genus level if applicable. In case no genus or family could be identified, we took the lowest identified taxonomy.

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Additional file 8:

Table S6 - univariate test showing correlations between amount of calprotectin in cecal matter and gut bacteria. Marked groups are those negatively linked to calprotectin amount (Rho < 0). Cut-off values are P < 0.05 and q < 0.1.

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Additional file 9:

Table S7 - P-values of genetic and cage effect calculated from NMDS analysis at all taxonomic levels. The randomized test was limited to 104 permutations.

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Additional file 10:

Figure S3 - visualization of genetic and cage effects using distance-based redundancy analysis. Genetic as well as cage effects show a strong correlation to the mice microbiome, as visualized in the dbRDA at the (a) phylum and (b) genus levels. Samples are colored by genotype; cages are visualized by connecting lines between samples.

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Additional file 11:

Table S8 - variation partitioning taking into account genotype, cage, and enterotype as well as shared information between these and unexplained variation. Percentage of variation in microbiota composition explained by solely genotype, cage and enterotype or by shared effects of those variables.

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Additional file 12:

Figure S4 - intra-strain dispersion of investigated mouse genotypes. Intra-strain dispersion was not significantly different between investigated genotypes, as shown here for genus level.

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Additional file 13:

Table S9 - PERMANOVA post hoc testing for significant differences of gut microbiota compositions between the five strains used. PERMANOVA post hoc testing for significant differences of gut microbiota compositions between the five strains used. The marked values are significant (P < 0.05, q < 0.1).

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Additional file 14:

Figure S5 - richness estimates at the OTU level over study factors. OTU richness estimated with a Chao1 estimator. (a) For genotypes significant differences in richness were observed. (b) Cage effect did not show any significant differences.

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Additional file 15:

Table S10 - list of taxa showing significant differences between genotypes. List of taxa showing significant differences between genotypes (stratified for ET1). Cut-off values of P < 0.05 and q < 0.1 were applied. The direction column sorts genotypes by their median abundance, from largest to smallest. A post hoc test was applied to direct neighbors in this list, where ' > ' is q-value of the test < 0.1, ' > > ' is q < 0.05 and ' > > > ' is q < 0.01.

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Additional file 16:

Table S11 - average abundance of bacterial groups showing significant differences between mouse genotypes. Values in brackets are standard deviations within the corresponding groups.

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Additional file 17:

Table S12 - bacterial groups showing significant differences between cages. Summary of bacterial groups showing significant differences between cages (P < 0.05 and q < 0.1). Male mice were excluded from this test. The direction column sorts genotypes by their median abundance from largest to smallest. A post hoc test was applied to direct neighbors in this list, where '=' is q-value > 0.1.

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Additional file 18:

Table S13 - PERMANOVA tests for community differences between genotypes and cages after removal of all Helicobacter OTUs.

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Additional file 19:

Table S14 - blocked Kruskal-Wallis test on all samples. (a) Blocked Kruskal-Wallis test on all (60) samples with enterotype as confounding factor yielded similar results, that is, bacterial groups showing significant difference between a) genotypes and b) cages as if enterotype had been pre-stratified.

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Additional file 20:

Figure S6 - schematic presentation of primer design used in the amplification of the V3-V5 region of 16SrDNA in this study.

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Additional file 21:

Table S15 - metadata of all mice used in the study, the OTU abundance of all samples and the OTU taxonomical assignments.

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