The ultra PAGE purified primers were ordered from Sangon, China. For each sample, one tube of PCR was performed. The PCR cycle condition was an initial denaturation at 94°C for 2 min; 25 or 30 cycles of 94°C 30 s, 57°C 30 S and 72°C 30S; and a final extention at 72°C for 5 min. The template dilution fold, the cycle number and the polymerase used were as listed in the table 1. For A, B, C, and D groups, each

20 μl reaction consisted of 2 μl Takara 10× Ex Taq Buffer (Mg2+ plus), 2 μl dNTP Mix (2.5 mM each), 0.5 μl Takara Ex Taq DNA polymerase (2.5 units), 1 μl template DNA, 1 μl 10 μM barcoded primer 967F, 1 μl 10 μM primer 1406R, and 12.5 μl ddH2O. For condition E, each 20 μl reaction consisted of 10 μl PfuUltra II Hotstart 2× Master

Mix, 1 μl template DNA, 1 μl 10 μM barcoded primer 967F, Epacadostat cost 1 μl 10 μM primer 1406R, and 7 μl ddH2O. Deep sequencing using Solexa GAII Barcode tagged 16 S V6 PCR products were pooled, purified (QIAquick PCR purification Kit, Qiagen), end repaired, A-tailed and pair-end adaptor ligated (Pair-end library preparation kit, Illumina). After the ligation of the adaptors, the sample was purified and dissolved in 30 μl of elution buffer, and 1 μl was then used as template for 12 cycles of PCR this website amplification. The PCR product was gel purified (QIAquick gel extraction kit, Qiagen) and directly sequenced using the 75 bp pair-end strategy on the Solexa GA II following the manufacturer’s instructions. The base-calling pipeline (version SolexaPipeline-0.3) was used to process the raw fluorescent images and the call sequences. Data MycoClean Mycoplasma Removal Kit analysis The paired-end reads were overlapped to assemble the final VRT752271 clinical trial sequence of V6 tags. The

sequencing quality of the Solexa platform decreases near the 3′ end. We used the first 60 bp from the 5′ end of each read for overlapping assembly. A pair was connected with a minimum overlap length of 5 bp and 0 mismatches in the overlapped region. We further trimmed all tags with any mismatches within primers, with any N bases or less than 35 bp for the V6 regions. The final high quality tags were allocated to each sample according to the barcode sequence. We performed taxonomic classification by assigning the reads of each sample to the 16 S V6 region database refhvr_V6 and then calculated the Global Alignment for Sequence Taxonomy (GAST) distance [27] (blastn release:2.2.18, e-value <1e-5, -b 50, http://​vamps.​mbl.​edu/​resources/​databases.​php). The OTU, rarefaction, Chao1 and ACE estimation were analyzed using the mothur (v.1.6.0, http://​www.​mothur.​org/​wiki/​Main_​Page) [18]. We wrote a Perl script to calculate the unique sequences (tags) and their abundance information for analyzing the rank-abundance curve of top abundant tags. The principal component analysis (PCA) was performed using Canoco (Version 4.51). The clustering analysis was performed using Primer 6.0.