Amplification and Sequencing of 16S rDNA

Genomic DNA was extracted as mentioned above, and the samples were identical to metagenomic sequencing. The V3-V4 variable region of the 16S rDNA gene was amplified using a previously described method (Gu et al., 2021 (link)). In general, PCR was performed using the KAPA HiFi Hot Start kit (Kapa Biosystems, Wilmington, MA, USA). The amplification procedure has been previously described (Liao et al., 2019 (link)). The AMPure XP beads (Beckman Coulter, Brea, CA, United States) was used to purify and quantify PCR products. The Illumina Nextera barcodes was added by secondary PCR procedure, and then the products were purified again to clear the nontarget fragments. The normalized and pooled amplicons were used for sequencing by using an Illumina NovaSeq 6000 system (Illumina, San Diego, CA, USA).