The UCLUST method [9] was used to cluster the filtered sequences with ≥97% similarity into Operational Taxonomic Unit (OTUs). Chimeric sequences were identified by ChimeraSlayer [10] and removed. Representative sequences

from each OTU were assigned Selleck CBL-0137 taxonomy using the Ribosomal Database Project classifier method [11] and the IMG/GG GreenGenes database of microbial genomes. A phylogenetic tree was constructed by applying the FastTree method [12] to the representative sequences. Rarefactions of 10 to 8,414 [minimum-maximum sequence depth] randomly selected sequences from each sample were used to calculate the Shannon index, a measure of within sample diversity, and to generate rarefaction plots. Pairwise comparisons of Shannon indices by subject and storage condition were obtained by Monte Carlo permutation. All p-values were adjusted by Bonferroni correction. To measure the diversity among subjects or storage conditions, a single rarefaction was performed at a sequencing depth of 4000 so that all samples were included in analyses. Distance matrices containing all pairwise comparisons were created for unweighted (presence/absence) dissimilarity values using the UniFrac Selleckchem GSK690693 phylogenetic method [13]. Principal coordinates were computed for the unweighted distance matrices and used to generate Principal Coordinate Analysis plots (PCoA). The non-parametric method, adonis [14], was used to identify significant

differences in phylogenetic distance variation by subjects and by storage condition. The Unweighted Pair Group Method with Arithmetic Mean (UPGMA) for clustering of samples was also carried out on the unweighted distance matrices [8]. A two-sample t-test was used to test for differences between the within and between group variances, with p-values adjusted by Bonferroni correction. Relative abundances of the three major phyla (Bacteroidetes, Firmicutes, Actinobacteria) were compared for the four methods, using the Mann–Whitney-Wilcoxon test, and compared by subject, using the Kruskal-Wallis test (SAS, version 9.3, SAS

Institute, Cary, NC). Results DNA from 24 fecal aliquots was successfully extracted and amplified. The OD 260/280 ratio, a measure of DNA purity, was greater than 1.8 in samples collected from card, D-malate dehydrogenase room temperature, and frozen methods; DNA purity from these methods were higher than DNA purity from RNAlater (Table  1, p < 0.05). From the initial 584,367 microbial 16S rRNA sequences, 347,795 sequence reads passed filtering criteria. 16.6% of these sequences were chimeric and subsequently removed resulting in 290,110 high-quality sequence reads (12,088 ± 7,302 [mean ± SD] sequences per sample) binned into one of 5,605 OTUs. The number of sequence reads did not differ significantly according to collection methods (Table  1, p = 0.84). Table 1 DNA purity and 16 s rRNA sequence reads by fecal collection method Methoda OD 260/280 (Mean ± SD)b Filtered sequence reads (Mean ± SD)d Method 1: Card 1.86 ± 0.