Briefly, the 16S rRNA amplicons and a mixture of amplicons at known concentrations were combined, fragmented using DNAseI (Invitrogen, Carlsbad, CA), and biotin-labeled using the recommended protocol for Affymetrix Prokaryotic Arrays. Labeled products were hybridized overnight at 48°C and 60 rpm. The arrays were washed, stained, and scanned as described in Hazen et al. [21]. Data collection signaling pathway and analysis Details on probe selection, probe scoring, data acquisition, and preliminary data analysis are presented in Hazen

et al. [21] and the analyses were performed by Second Genome (San Bruno, CA, USA). In brief, two criteria were met when the probe pairs scored as positive: (i) the PM (Perfect Match) probe’s intensity of fluorescence was greater than 1.3 times that from the MM (Mismatch) JNK-IN-8 cell line control and (ii) the difference in intensity, PM minus MM, was at least 500 times greater than the squared noise value (>500 N 2), which was the variation in pixel intensity signals observed by the scanner as it read the array surface. An OTU was considered present in the sample when over 90% of its assigned probe pairs were positive. A hybridization intensity score (HybScore) was calculated in arbitrary units for each probe set as the trimmed average (maximum and minimum values removed

before averaging) of the PM minus MM intensity differences across the probe pairs in a given probe set. The values Demeclocycline of the present OTUs used for each taxa-sample intersection were populated in two distinct ways. In the first case, the abundance metrics were used directly (AT). In the second case, RGFP966 supplier binary metrics were created where 1’s represented presence, 0′s indicated absence (BT). OTUs were filtered

in several different manners. Filter-1 includes OTUs present in at least one of the samples. Filter-3 includes OTUs present in samples from one treatment but not detected in any samples of the other treatments. Filter-5 includes OTUs whose abundance significantly increased in one treatment compared to the other treatments and Filter-9 includes OTUs with unique abundance patterns within a species. For Filter-3, the percent prevalence required among the samples in one state began at 100% but then decreased until the OTU set intersected all samples. Thus, each sample contained a present call for at least one of the passing OTUs. The Unifrac distance metric determines the dissimilarity between communities by using the phylogenetic distances between OTUs [34]. For the weighted Unifrac distance metric, WUnifrac, the OTU abundance was also considered. The presence/absence (BT) data, used Unifrac; whereas, the abundance data (AT) used WUnifrac. For Filter-5, p-values were calculated using the parametric Welch test. In this exploratory analysis, false discovery rates were not considered in the p-value calculations.