66 dnadist and neighbour [52]. The tree was visualized with MEGA4 [57]. A phylogenetic tree was constructed for the OTU representatives of the phylum Actinobacteria. For Bifidobacteriales and Actinomycetales, sequences with nearest FASTA EMBL Prokaryote search (all >98% similarity), and for Coriobacteriales sequences with nearest FASTA EMBL prokaryote and environmental selleck chemicals database searches (>85% and >91%, respectively), were selected and aligned together with OTU representative sequences.
Sequences from the European ribosomal RNA database representing Actinobacteria and Clostridium leptum (AF262239) were used as a reference in the profile alignment (Additional file 4). The alignment, distance matrix, and visualizing was done as described above. A bootstrap analysis of hundred replicates was performed using seqboot and consense programs selleck screening library of Phylip 3.66 [52]. To describe whether the phylogenies of the combined sequence data from the fractioned libraries
and the unfractioned library were significantly different, the UniFrac Significance analysis was applied for each pair of environments using abundance weights [58]. The UniFrac Lineage-specific analysis was used to break the tree up into the lineages at a specified distance from the root, and to test whether any Eltanexor molecular weight particular group differed between the sample libraries [58]. The phylogenetic tree for the analyses was constructed from OTU representative sequences determined separately for
the combined fractioned libraries and for the unfractioned library as described above, with the exception that in the profile alignment a root sequence (Methanobrevibacter smithii AF054208) was added and left to the alignment. Comparison of individual libraries using SONS The microbial community composition Ergoloid differences between libraries of individual %G+C profile fractions and the unfractioned sample were analysed using SONS [24], which calculates the fraction of sequences observed in shared OTUs in each library (Uobs and Vobs) and the observed fraction of shared OTUs in each library (Aotu_shared and Botu_shared). For the SONS analyses, an alignment with all of the sequences from the clone libraries of the fractioned sample and the unfractioned sample was created, and a distance matrix was calculated as described above in the Sequence analysis and alignment section. Shannon entropies of clone libraries of the %G+C profiled sample To compare the diversity of the clone libraries derived from the fractioned sample, OTUs were also determined using a Bayesian clustering method [59], followed by the estimation of Shannon entropies with a standard Bayesian multinomial-Dirichlet model. In the estimation, 100 000 Monte Carlo samples were used for each library under a uniform Dirichlet prior [60]. The Shannon entropy value correlates with the amount and evenness of clusters or phylotypes in a community sample, but disregards the disparity between them [61].