title: 16S rRNA of soil
design: The sequence containing the variable V3 and V4 regions was amplified using the forward primer (338F 5'- ACTCCTACGGGAGGCAGCA-3') and the reverse primer (806R5'- GGACTACHVGGGTWTCTAAT-3'). The PCR process was performed according to Zhao et al. (2016), with slight modification. The programs were as follows: 1 cycle at 95C for 5 min; 25 cycles at 95C for 30 s, 50C for 30 s, 72C for 40 s; 1 cycle at 72C for 7 min, and a final soak at 10C. The PCR reactions were performed in a 50 L mixture containing 40 ng of genomic DNA, 1.5 L of bar-PCR primer F (10 M), 1.5 L of primer R (10 M), 0.2 L of Q5 high-fidelity DNA polymerase, and 10 L of high-GC enhancer, 10 L of PCR buffer, and 1 L of dNTPs (10 mM). Water was added to keep the final volume at 50 L. The size and purity of amplicons were checked by electrophoresis on a 1.8% agarose gel. The short, non-specific amplification products were removed with AMP beads according to the manufacturers protocol. The concentration of purified DNA was measured using NanoDrop 2000, and the amplicons were mixed at a 1:1 ratio and shaken well. Sequencing of PCR products was performed on the Illumina HiSeq2500 platform ( Biomarker Technology, Ltd. Beijing, China)(Joshi et al., 2017).