The 16S rRNA gene in fecal microbiota and shotgun metagenomic sequencing.
Fresh fecal samples were collected and frozen at −80°C until DNA extraction. According to the manufacturer's protocol, total fecal DNA was extracted from 0.25 g of each homogenized sample using the QIAamp PowerFecal DNA kit (Qiagen). The DNA yield and quality were then checked with a Multiskan GO spectrophotometer (Thermo Fisher Scientific, USA). To identify bacterial communities, 16S rRNA gene V3-V4 amplicon libraries were generated in a 20-μL reaction mixture containing 10 μL KAPA HiFi HotStart ReadyMix (KAPA Biosystems, USA), 2 μL DNA (60 ng), and 1 μL of each of the barcoded primers (10 μM). The forward and reverse primers used were 5′-