16S rRNA Sequencing Library Preparation

The 16S rRNA sequencing library was constructed according to the Illumina 16S Metagenomic Sequencing Library Preparation protocol (Illumina) targeting the V3 and V4 hypervariable regions of the 16S rRNA genes using primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′)^{16 (link)}. MightyAmp DNA Polymerase v. 2 (Takara Bio Inc.) was used for the PCRs. The initial PCR was performed using region-specific primers to ensure compatibility with the Illumina index and sequencing multiplex adapters. The amplified fragments were separated on 2% agarose gels, stained with Safelook Load-Green (Wako), and visualized on the FAS Nano Gel Document System (Nippon Genetics). The amount of purified DNA recovered was quantified using a Spectro/Fluorometer (DS-11FX+, DeNovix). An equimolar mixture of all PCR products was sent to a commercial company for 2 × 300 bp paired-end sequencing on the MiSeq platform using Illumina MiSeq v3 Reagent Kit (Fasmac, Kanagawa, Japan).