B.C.U.C.B
Several forward bacterial primers from the Ion 16S Metagenomics kit (Life Technologies, United States) were used to amplify the 16S rRNA genes targeting the V2 to V9 variable regions. A total reaction mixture of 30 uL 2x Ion 16S Metagenomics Environmental Master Mix (Life Technologies, United States), 30 ng DNA template, 0.25 uM of each primer, and sterile dH2O was used for PCR amplification. The amplification started at 95C for 10 min, followed by 25 cycles of 95C for 30 s, 58C for 30 s, and 72C for 20 s each, and 72C for 7 min. The presence of PCR amplicons was verified on a 1% EtBr-stained agarose gel containing 1 TAE buffer. The purification of PCR amplicons, template preparation for the sequencing library, and chip loading were performed following the user guide of the Ion 16S Metagenomics kit. In brief, the Agencourt AMPure XP Reagent (Beckman Coulter, United States) was used to purify PCR amplicons, and the concentration was determined thereafter using the Agilent High Sensitivity DNA Kit (Agilent Technologies, United States) and the Agilent 2100 Bioanalyzer. The PCR amplicons were diluted to approximately 25 pM and the templates for the sequencing library were prepared with the Ion Personal Genome MachineTM (PGMTM) Hi-QTM View OT2 Kit (Life Technologies, United States), after which emulsification PCR was run on the Ion OneTouchTM 2 System. Finally, the products were loaded onto the Ion 318TM Chip v2 using the Ion PGMTM Hi-QTM Sequencing Kit (Life Technologies, United States) and PGMTM System for sequencing.