We tested the difference between

pairs using distance based NP-MANOVA, which yielded p = 0.085 for unweighted UniFrac and p = 0.197 for weighted UniFrac. Thus the two gold standards were not significantly different. Figure 3A shows the unweighted UniFrac analysis colored to distinguish communities from the 10 individuals studied. Figure 3B shows the same scatter plot colored by storage method, and Figure 3C shows the plot colored by extraction method. The data emphasizes that individuals differ substantially from Cyclosporin A chemical structure each other, and that storage and extraction methods have less pronounced effects. Also present in each individual cluster are the two replicates from 1 cm apart, emphasizing the reproducibility of

the method. Statistical analysis was carried out by asking whether unweighted UniFrac distances were greater within groups than between groups, then 10,000 label permutations were used to generate an empirical P-value. Clustering by subject was highly significant (P < 0.0001). No significance was seen for clustering by extraction check details method (P = 0.16) or storage method (P = 0.98). We conclude that overall clustering, when analyzed for presence or absence of different bacterial groups, is dominated by differences between individuals. Figure 4 shows the weighted UniFrac analysis, which takes into account information on relative abundance, comparing the influence of individual of origin (Figure 4A), extraction method (Figure 4B), or storage method (Figure 4C). Again the differences among subjects were highly significant (P < 0.0001), but now the differences due to extraction methods were also significant (P = 0.001). Differences due to storage method were not significant. Thus when the proportional

representation of different taxa is taken in to account, both Megestrol Acetate the subject of origin and the extraction method exert significant effects. We next investigated whether significant clustering could be detected when each extraction method was compared individually to the collection of other extraction methods. Again UniFrac distances were analyzed for within group and between group comparisons, and an empirical P-value generated from 10,000 permutations. No significant clustering was seen in the unweighted analysis. However, using weighted UniFrac significant clustering was seen for the phenol-bead beating method (P = 0.041) and the Qiagen method (P = 0.0014). The strong effect of the Qiagen method was driven in part by the fact that the most samples were analyzed using the Qiagen method, so the sample size was relatively large. Comparison of each method to the two gold standards using Selleck Fludarabine NP-MANOVA showed that the phenol bead beating and PSP methods both achieved p = 0.001.