2.3.2. PCR amplification of 16S rRNA genes and Miseq sequencing

Polymerase chain reaction (PCR) amplification of 16S rRNA genes was performed using general bacterial primers (515F 5'‐GTGCCAGCMGCCGCGGTAA‐3' and 926R 5'‐CCGTCAATTCMT TTGAGTTT‐3'). The primers also contained the Illumina 5'overhang adapter sequences for two‐step amplicon library building, following manufacturer's instructions for the overhang sequences. The initial PCRs were carried out in 25 μl reaction volumes with 1‐2μl DNA template, 250 mM dNTPs, 0.25 mM of each primer, 1× reaction buffer, and 0.5 U Phusion DNA Polymerase (New England Biolabs). PCR conditions consisted of initial denaturation at 94°C for 2 min, followed by 25 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s, with a final extension of 72°C for 5 min. The second step PCR with dual 8‐base barcodes was used for multiplexing. Eight cycle PCR reactions were used to incorporate two unique barcodes at either end of the 16S amplicons. Cycling conditions consisted of one cycle of 94°C for 3 min, followed by eight cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 30 s, followed by a final extension cycle of 72°C for 5 min. Prior to library pooling, the barcoded PCR products were purified using a DNA gel extraction kit (Axygen) and quantified using the FTC ‐3000 TM real‐time PCR. The libraries were sequenced by 2*300 bp paired‐end sequencing on the MiSeq platform using MiSeq v3 Reagent Kit (Illumina) at Tiny Gene Bio‐Tech (Shanghai) Co., Ltd.