Bacterial diversity revealed by 16S rRNA gene pyro-tags

16S rRNA genes targeting V3 and V4 regions were amplified from the triplicate DNA extracts of each sample following the procedures specified before [38]. PCR amplicons were purified with a quick-spin Kit (iNtRON, Seoul, Korea), and concentrations were measured by NanoDrop^® spectrophotometer ND-1000. The purified amplicons were sent out for pyrosequencing on the Roche 454 FLX Titanium platform at the BGI Company (BGI, Shenzhen, China).

Raw reads were analyzed using Quantitative Insights Into Microbial Ecology (QIIME v. 1.3.0) pipeline [41]. The sequencing data were initially de-multiplexed and separated into different samples based on their nucleotide barcodes. Then, the sequences in each sample were denoised by AmpliconNoise. Chimera checking was performed using Chimera Slayer [42]. The effective reads which we called pyro-tags were produced. Briefly, the pyro-tags were clustered and assigned to operational taxonomic units (OTUs) with 97% similarity cutoff. Taxonomy of representative sequences from each OTU was aligned using the SILVA bacterial database implemented in Mothur platform (v. 1.33.2) [43]. And the diversity indicators including Good’s coverage, ACE and Chao 1 richness estimators were calculated as well.