2.5 | PCR amplification and MiSeq high-throughput
sequencing
Bacterial 16S rRNA was amplified using the 338F primer set
(5′-ACTCCTACGGGAGGCAGCA-3′) and 806 R (5′-GGACTACHVGGGTWTCTAAT-3′)( Chen
et al., 2018). The reaction mixtures (20 μL) contained 5 × FastPfu
Buffer (4 μL), 2.5 mM dNTPs (2 μL), 5 μM forward primer (0.8 μL), 5 μM
reverse primer (0.8 μL), FastPfu polymerase (0.4 μL), and template DNA
(10 ng). The reaction volume was made up to 20 μL with ddH2O. The
thermocycler settings were as follows: 2 min at 95 ℃, followed by 27
cycles for 30 s at 95 °C, 2 min at 45 °C, 3 min at 72 °C, and finally 10
min at 72 °C. Amplified products were visualized using 2% agarose gel
and purified followed the manufacturer’s protocols for the AxyPrep DNA
Gel Extraction Kit (Axygen Biosciences, USA) and quantified using
QuantiFluor™-ST (Promega, USA). Sequencing was performed using the
Illumina MiSeq platform (Illumina, San Diego, CA, USA) in Majorbio
Bio-Pharm Technology Co. Ltd. (Shanghai, China).