16S rRNA amplicon sequencing of human stool
The 16S rRNA primer pair 341F (5-CCTACGGGNGGCWGCAG-3) and 785R (5-GACTACHVGGGTATCTAATCC-3) was utilized to evaluate the microbial ecology of each sample on the MiSeq platform with methods via the bTEFAP DNA analysis service. Each sample underwent a single-step 30 cycle PCR using HotStarTaq Plus Master Mix Kit (Qiagen, Hilden, Germany) under the following conditions: 94 C for 3 minutes, followed by 30 cycles of 94 C for 30 seconds, 53 C for 40 seconds, and 72 C for 1 minute, after which a final elongation step at 72 C for 5 minutes was performed. Following PCR, all amplicon products from different samples were mixed in equal concentrations and purified using Agencourt Ampure beads (Agencourt Bioscience, Beverly, MA). Samples were sequenced utilizing the Illumina MiSeq chemistry following manufacturers protocols (Illumina, San Diego, CA).