2.6. Next-Generation Sequencing (NGS) Analysis
Following DNA extraction and purification, the extracted DNA served as the template for PCR amplification. The target region for amplification was the V3-V4 region of the bacterial 16S rRNA gene, using the primer pair 314F (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′) and 805R (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGTATCTAATCC-3′). The amplification was performed with KAPA HiFi HotStart ReadyMix (Roche Sequencing Solutions, Pleasanton, CA, USA [KK2601]), using thermal cycling conditions of 95 °C for 5 min, 30 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, and final extension at 72 °C for 5 min. Next, DNA libraries were constructed by ligating Nextera XT Index and Illumina sequencing adapters to the PCR products. The prepared libraries were subjected to paired-end sequencing (2 × 300 bp) on the Illumina MiSeq platform (Illumina, San Diego, CA, USA) by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China), to generate the necessary sequencing data for subsequent analysis.