2.4.2. SMRT Sequencing of Bacterial Diversity

The full-length 16S ribosomal RNA (rRNA) gene was amplified by PCR for SMRT sequencing using the forward primers 27F (5′-AGRGTTTGATYNTGGCTCAG-3′) and the reverse 1492R (5′-TASGGHTACCTTGTTASGACTT-3′). The PCR program was as follows: 95 °C for 5 min; 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 90 s, with a final extension of 72 °C for 7 min. Sequence pre-processing were performed on a PacBio Sequel platform (Pacific Biosciences, Menlo Park, CA, USA). Raw Barcode-CCS sequence data was obtained with lima v1.7.0 software. Then filtering raw Barcode-CCS to get valid sequence. Using the Quantitative Insights into Microbial Ecology (QIIME) package (version 4.2) to remove the low-quality sequence. The unique sequence set was classified into OTUs based on a 97% threshold identity using UCLUST [19]. Subsequently, representative sequence was compared using the Mothur3 software with the Silva database to gain classified information [20]. Functional genes of the bacterial communities were predicted using phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) [21]. The final predicted metagenome was analyzed by Statistical Analysis of Taxonomic and Functional Profiles (STAMP) v.2.1.3 [22] Cluster analysis produced using R software (ver. 3.2.5) based on the OTUs. Prior to the redundancy analysis (RDA) was analyzed using detrended correspondence analyses that were conducted by the R software package4 (ver. 3.2.5) [23].