Bacterial 16S rDNA Amplicon Sequencing Protocol

During the polymerase chain reaction, which was conducted with a TopTaq DNA polymerase kit (Transgen, Beijing, China), the V3–V4 regions from the bacterial 16S rDNA gene were amplified with universal primer set 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) [41 (link)]. According to the manufacturer’s protocol, final PCR products were purified and quantified by Agencourt AMPureXPPCR Purification Beads (Beckman Coulter, Grants Pass, OR, USA) and Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The sequencing was performed on Illumina NovaSeq 6000 platform (2 × 250 bp) at Genesky Biotechnologies Inc. (Shanghai, China). Amplicon sequencing is the high-throughput sequencing of PCR products of specific gene segments such as 16S rDNA, 18S rDNA, and ITS [42 (link)]. Among them, the 16S rDNA sequencing used in this study is an important and widely used tool for obtaining information about microbial communities’ structures, microbial taxonomy, and community diversity [43 (link)].