Bacterial 16S rDNA Gene Amplification

PCR was conducted using a TopTaq DNA polymerase kit (Transgen, China) to amplify the V3-V4 region from the bacterial 16S rDNA gene with the primer sets Primer F (Illumina adapter sequence 1 + CCTACGGGNGGCWGCAG) and Primer R (Illumina adapter sequence 2 + GACTACHVGGGTATCTAATCC). Three 1-μL aliquots from each DNA sample were used as a template for amplification according to the manufacturer’s protocols. Illumina index sequences were specifically designed to identify each DNA sample using the following barcoded primers: Fwd 5′-AATGATACGGCGACCACCGAGATCTACACXXXXXXXXACACTCTTTCCCTACACGACGCTCTTCCGATCTCTG-3′ and Rev 5′-CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAC-3′, in which X indicates a unique barcode for each sample. The amplified products were checked by agarose gel electrophoresis and purified with magnetic beads. All PCR products were sequenced by MiSeq reagent kit version 3 (Illumina, San Diego, CA, USA). Similarly, detailed sequencing methods can be found in the following literature [25 (link),38 (link),39 (link),40 (link),41 (link),42 (link)].