The phylogenetically informative V3-V4 region of the 16S rRNA (rRNA) gene was amplified using universal primer 347F/803R (66 (link), 67 (link)). The dual-barcoding approach was applied as previously described (68 (link), 69 (link)) to label the 16S rRNA gene amplicons of each sample. Briefly, the 6-mer barcodes were attached on the 5′ ends of both forward and reverse PCR primers such that the 16S rRNA gene PCR amplicons from each sample contained a unique dual-barcode combination. The PCR primers were synthesized by Sangon Biotech, Shanghai, China, and the primer sequences are shown in Table S1 in the supplemental material. The 25-μl PCR mixture contained 300 ng of sample DNA as a PCR template, 1 μl of 10 μM forward and reverse 16S primers, and 12.5 μl of 2× HotMaster Taq DNA mix (Tiangen Biotech, Beijing, China). The PCR was performed on an Applied Biosystems model 2720 thermal cycler (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 94°C for 3 min and then at 94°C for 30 s, 58°C for 30 s, and 72°C for 20 s for 30 cycles followed by 72°C for 4 min. The integrity of the PCR products was verified by agarose gel electrophoresis. After a purification step was performed with a gel purification kit (Promega, Madison, WI, USA), the 16S PCR amplicons were pooled at equal levels of molarity, freeze-dried, and submitted to the New York Genome Center for sequencing.
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