Amplification and Sequencing of 16S rDNA

The V1–V3 region of the 16S rDNA gene was amplified with barcoded primers (8F/533R) (Wu et al., 2012 (link)). Replicated PCR products of each sample were pooled and separated by 2% agarose gel electrophoresis, then purified with a DNA gel extraction kit (Axygen, China). Prior to sequencing, the concentrations of purified amplicon DNA were determined using a QuantiFluorTM-ST Fluorometer (Promega, Madison, WI, United States). Equal amounts of the PCR products were mixed together and sequenced on a 454 GS FLX + platform (Roche Applied Science, Indianapolis, IN, United States) at Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China.