K_08_2
The V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified using the primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). PCR amplification was performed in a 20 L reaction system containing 4 L of 5 FastPfu Buffer, 2 L of 2.5 mM dNTPs, 0.8 L of each primer (5 M), 0.4 L of FastPfu Polymerase, 0.2 L of BSA, and 10 ng of template DNA. The PCR conditions were as follows: 95C for 3 min, followed by 27 cycles of 95C for 30 s, 55C for 30 s, and 72C for 45 s, with a final extension at 72C for 10 min. The amplicons were purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using QuantiFluor-ST (Promega, USA). The purified amplicons were pooled in equimolar concentrations and paired-end sequenced (2  300 bp) on the Illumina NextSeq 2000 platform according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).