8% agarose gel and a QIAquick Gel Extraction Kit (Cat# 28704, Qiagen) per the manufacturer’s instructions. Defined DNA community composition Two defined DNA mixture were created using 10 different plasmids, each containing a near full length 16S rDNA amplicon, obtained using primers BSF8 and BSR1541. One mixture had an equal amount of each plasmid and one was staggered to contain different proportions of each clone. The strains and proportions on the Staggered mix are: Clostridium dificile (ATCC#: BAA-1382) – 39.99%, Bacteroides fragilis (ATCC#: 25285) – 32.01%, Streptococcus pneumoniae (ATCC#: BAA_334)
– 4.92%, Desulfovibrio vulgaris (ATCC#: 29579) – 1.95%, Campylobacter jejunii (ATCC#: 700819) – 2.03%, Rhizobium vitis (ATCC#: BAA_846) – 2.00%, Lactobacillus Selleckchem PND-1186 delbruekii (ATCC#: BAA-365) – 5.06%, Escherichia coli HB101 – 2.01%, Treponema sp. (macaque stool clone) – 7.97%, and Nitrosomonas sp. (environmental clone) – 2.04%. Clones were made using the Topo-XL kit (Cat# K4700-20, Invitrogen, Carlsbad, CA). Two polymerases were tested for the Staggered mix, AmpliTaq (as used for stool DNA samples) and GreenTaq (Promega, Madison,
WI) as per manufacturer instructions. The PCR cycling conditions were the same as described for the stool sample DNA. 454/Roche sequencing methods Purified amplicon DNAs were quantified using Quant-iT PicoGreen kit (cat# P7589, Invitrogen, Carlsbad, CA) and pooled for pyrosequencing. Pyrosequencing using the 454/Roche GS FLX chemistry was carried out according to the manufacturer’s instructions. Pyrosequencing using the Titanium MK-8931 method was carried out using the Titanium genomic kit. Primers for PCR amplification MLN2238 datasheet of rDNA gene segments are in Additional File 3. The rDNA region amplified with V1-V2 primers used for FLX sequencing is contained within
the regions amplified with the V1-V3 primers used for Titanium sequencing. Pyrosequence reads were uploaded into QIIME and processed as described (Caporaso et al., 2010). Briefly, QIIME accepts as input bar coded 16S rRNA gene sequences, classifies them using the RDP classifier , aligns them using Pynast , constructs phylogenetic trees using FastTree2, calculates UniFrac distances, and generates data summaries of the proportions of taxa present and PCoA plots based on UniFrac distances. We used 97% OTUs in the analysis. For the RDP very classifier, we required >50% confidence for all calls. Accession numbers for sequences determined here are in Additional File 5. Statistical methods Clinical characteristics were compared as median, range, counts and percentages. For analysis in Figures 1 and 2, no corrections for multiple comparisons were applied. UniFrac [33, 34, 41] was used to generate distances between all pairs of communities; both weighted and unweighted UniFrac were used in the analyses. Statistical analysis was carried out by comparing distances within groups to distances between groups.