16S metagenomic community of Mixed-b-3d
Total DNA was extracted from cell pellets using EZNATM Mag-Bind Soil DNA Kit (Omega Biotek, USA) according to instruction manual. The DNA concentration and quality were determined by NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, USA). Qualified DNA samples were sent to Sangon Biotech Shanghai Co.Ltd (Shanghai, China) for sequencing and bacterial community analysis. Two rounds PCR were performed to amplify the bacteria 16S rRNA genes. First round PCR was amplified with primers 341F (5-CCTACGGGNGGCWGCAG-3) and 805R (5-GACTACHVGGGTATCTAATCC-3) spanning the V3-V4 hypervariable region of the 16S rRNA gene. PCR products from first round PCR were used as templates for second round PCR with Illumina Nested primers. To construct the PCR amplicon libraries, PCR products from second round PCR were normalized in equimolar amounts before sequencing. Sequences, which obtained from Illumina HiSeq 2000 (Illumina, San Diego, USA), were phylogenetically allocated down to the genus level with the RDP classifier by Nave Bayesian assignment algorithm.