Cleanpcr

The PCR amplification and sequencing were done by Chunlab, Inc. (Seoul, Korea). Briefly, PCR amplification was performed using primers targeting the V3–V4 hypervariable regions of the 16S rRNA gene with extracted DNA, using primers 341F (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGG CWGCAG-3′; and 805R (5′-GTCTCGTGGGCTCGG-AGATGTGTATAAGAGACAGGACT ACHVGGGTATCTAATCC-3′). Then, secondary amplification was carried out using a unique dual index with i5 forward primer (5′-AATGATACGGCGACCACCGA GATCTACAC-XXXXXXXXTCGTCGGCAGCGTC-3′; and i7 reverse primer (5′-CAAGCAGAAGACGGC ATACGAGATXXXXXXXX-GTCTCGTGGGCTCGG-3′) (X indicates the barcode region) to attach the Illumina NexTera barcode. The PCR product was confirmed using 1% agarose gel electrophoresis, visualized under a Gel Doc system (BioRad, Hercules, CA, USA), then purified and removed non-target products with the CleanPCR (CleanNA). The quality and product size were determined on a Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA) using a DNA 7500 chip. Mixed amplicons were pooled, and the sequencing was performed at Chunlab, Inc. (Seoul, Korea), with the Illumina MiSeq Sequencing system (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions.

The V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified with the primers 341F (5′-TCGTCGGCAGCGTC-AGATGTGTATAAGAGACAG-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GTCTCGTGGGCTCGG-AGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3′). The thermal profile for the PCR was a cycle at 95 °C for 3 min and 25 cycles of 95 °C for the 30s, followed by 55 °C for 30s and 72 °C for 30s, and a final cycle at 72° for 5 min. Secondary amplification for attaching the Illumina NexTera barcode was performed with i5 forward primer (5′-AATGATACGGCGACCACCGAGATCTACAC-XXXXXXXX-TCGTCGGCAGCGTC-3′; X indicates the barcode region) and i7 reverse primer (5′-CAAGCAGAAGACGGCATACGAGAT-XXXXXXXX-GTCTCGTGGGCTCGG-3′). The condition of secondary amplification is the same as the former, except for the amplification cycle set to 8. The PCR product was confirmed by 1% agarose gel electrophoresis, visualized under a Gel Doc system (BioRad, Hercules, CA, USA), and purified with the CleanPCR (CleanNA, Waddinxveen, The Netherlands). The product quality and size were assessed on a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) using a DNA 7500 chip. The sequencing was carried out at Chunlab, Inc. (Seoul, Korea) with Illumina MiSeq Sequencing System (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions.