Profiling Gut Microbiome via 16S rRNA Sequencing

Bacterial DNA was extracted from feces using QIAamp DNA stool mini kits (Qiagen). PCR amplification was performed using primers targeting the segment from the V3 to V4 regions of the 16S rRNA gene with extracted DNA. For bacterial amplifications, we used barcoded primers of 341F (5′-CCTACGGGNBGCASCAG-3′) and 805R (5′-GACTACNVGGGTATCTAATCC-3′). The amplifications were performed under the following conditions: initial denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, primer annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds, with final elongation at 72°C for 5 minutes. The QIAquick PCR purification kit (Qiagen) was used to purify the purification of the amplified products. Equal concentrations of purified products were pooled, and short fragments (non-target products) were removed with an AMPure bead kit (Agencourt, Beverly, MA). The quality and product size were assessed on a Bioanalyzer 2100 (Agilent) using a DNA 7500 chip. Mixed amplicon sequencing was conducted by emulsion PCR and then deposited on picotiter plates. The sequencing was carried out at Chunlab (Seoul, South Korea) on a GS Junior Sequencing System (Roche, Basel, Switzerland) per the manufacturer’s instructions. Taxonomic cladogram was analyzed using LEfSe with a threshold 2 on the logarithmic LDA score.⁶¹ (link)