sequencing of bacterial 16S rRNA gene V3-V4 region
Microbial DNA was extracted according to the instructions of NucleoSpin Soil Kit (Macherey-Nagel, Germany). The V4 region of prokaryotic 16S rRNA genes was amplified using the primer pairs 338F (ACTCCTACGGGAGGCAGCAG) and 806R ( GGACTACHVGGGTWTCTAAT). PCR was performed using Phusion High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA). PCR cycling condition was as follows: 3 min denaturation at 95 C; 30 cycles of denaturation at 95 C for 45 s, annealing at 56 C for 45 s, elongation at 72C for 45 s; and final extension at 72 C for 10 min. The PCR products were used to construct libraries used for sequencing on MiSeq platform, according to the manufacturer's recommendations.