title: ITS rDNA
design: Total genomic DNA was extracted from samples using the TGuide S96 Magnetic Soil /Stool DNA Kit (Tiangen Biotech (Beijing) Co., Ltd.) according to manufacturers instructions. The quality and quantity of the extracted DNA were examined using electrophoresis on a 1.8% agarose gel,and DNA concentration and purity were determined with NanoDrop 2000 UV-Vis spectrophotometer (Thermo Scientific, Wilmington, USA).The full-length 16S rRNA gene were amplified with primer pairs 27F: AGRGTTTGATYNTGGCTCAG and 1492R: TASGGHTACCTTGTTASGACTT.Both the forward and reverse 16S primers were tailed with sample-specific PacBio barcode sequences to allow for multiplexed sequencing. We chose to use barcoded primers because this reduces chimera formation as compared to the alternative protocol in which primers are added in a second PCR reaction. The KOD One PCR Master Mix (TOYOBOLife Science) was used to perform 25 cycles of PCR amplification, with initial denaturation at 95 C for 2 min, followed by 25 cycles of denaturation at 98 C for 10 s, annealing at 55 C for 30 s, and extension at 72 C for 1 min 30 s, and a final step at 72 C for 2 min. The total of PCR amplicons were purified with VAHTSTM DNA Clean Beads (Vazyme, Nanjing,China) and quantified using the Qubit dsDNA HS Assay Kit and Qubit 3.0 Fluorometer (Invitrogen, Thermo Fisher Scientific, Oregon, USA). After the individual quantification step, amplicons were pooled in equal amounts. SMRTbell libraries were prepared from the amplified DNA by SMRTbell Express Template Prep Kit 2.0 according to the manufacturers instructions (Pacific Biosciences).