Profiling Gut Bacterial Diversity

The colonic bacterial DNA was isolated with the E.Z.N.A.^® soil kit (Omega Bio-tek, USA) according to the instructions. DNA concentration and purity were investigated by NanoDrop2000. The V3-V4 regions were amplified using specific primers, 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). PCR products were purified with AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, USA). Sequencing of the TruSeq DNA libraries was done on a Miseq PE300 sequencer (Illumina, USA) following the manufacturer’s recommendations. Trimmomatic software was used for quality control and FLASH (Fast Length Adjustment of SHort reads) software was used for splicing. The taxonomic unit was screened for further annotation (Korpela et al., 2016 (link)).