Illumina sequencing library construction and 16S V3-V4 metagenomic sequencing

For sequencing each bacterial 16S rRNA gene, the case process for constructing the Illumina 16S metagenomic sequencing library was conducted using the Illumina official protocol (Illumina, San Diego, CA, USA). The present study targeted the V3-V4 hypervariable region on the bacterial 16S rRNA gene for metagenomic sequencing. The region was amplified by polymerase chain reaction (PCR) using KAPA Hot Start Ready Mix (2X) (Roche, Basel, Switzerland). A pair of amplicon primers targeting the 16S V3-V4 region was used for PCR amplification as recommended by Illumina. After PCR amplification, PCR products were purified using an AMPure XP bead (Beckman Coulter, Pasadena, CA, USA). To construct the 16S metagenomic sequencing library, additional PCR amplification was performed using Nextera XT Index Kit (Illumina), which contained the Illumina multiplexing dual index and sequencing adapter. Thereafter, PCR products were purified again by using an AMPure XP bead. Each sequencing library was subjected to metagenomic sequencing following the paired-end 2 ×300 bp Illumina MiSeq™ protocol.