4.2.3. 16S V3-V4 Metagenome Sequencing
The 16S V3-V4 amplicon libraries for Next Generation Sequencing (NGS) based metagenome sequencing were prepared on the recommendation of the official Illumina 16S V3-V4 metagenome sequencing library preparation protocol, which targeted the V3-V4 hypervariable region within the bacterial 16S ribosomal RNA gene (Illumina, San Diego, CA, USA). All PCR amplification for sequencing library preparation was conducted using 2X KAPA HiFi Hot Start Ready Mix (Roche, Charles Avenue, UK). For this experimental purpose, the 16S V3-V4 hypervariable region-specific universal primer pair was used. The primer sequences were as follows:16S 341F forward primer is 5′ -TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′. 16S 806R reverse primer is 5′ -GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3′. After PCR amplification, all amplicon products were purified using the AMPure XP beads (Beckman Coulter, Brea, CA, USA). The additional PCR amplification was then performed to add the Illumina adapter and multiplex indices using the Nextera XD Index (Illumina, USA). The final PCR products were then purified once again using the AMPure XP beads. After the metagenome sequencing library preparation, the 16S V3-V4 metagenome sequencing was carried out using Illumina Miseq (2 × 300) paired-end sequencing workflow (Illumina, USA).