design: Based on concentration, amplicons were pooled in equimolar ratios and purified with the E.Z.N.A. Gel Extraction Kit (Omega Bio-tek, Norcross, GA, USA). The target DNA fragments were eluted in TE buffer, and the final DNA concentration was determined using a NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific). Only high-quality DNA samples proceeding to high-throughput sequencing for microbial community analysis.