16s rRNA of gut microbiota: adult
Total bacterial DNA was extracted using a Fecal DNA Bead Isolation kit (Bioeasy, Shenzhen) according to the manufacturers instructions on a KingFisher FLEX instrument for automatic processing. Identical protocols were followed for all samples, including the addition of three external standards to each batch. The 16 S rRNA gene V4 region was amplified by the following barcoded primers (shown from 5 to 3): V4F, GTGYCAGCMGCCGCGGTAA and V4R, GGACTACNVGGGTWTCTAAT. The barcodes were designed using Barcrawl so that there were 3-bp differences between any two barcodes. The following three PCR machines were used in the present study: ABI-1:ViiA 7 Dx, ABI-2:ViiA 7 and Bioneer. They were operated by two professional technicians. The following PCR conditions were used: initial denaturation at 94 C for 5 min followed by 30 cycles of denaturation at 94 C for 30 s, annealing at 52 C for 30 s, extension at 72 C for 45 seconds and a final extension at 72 C for 5 min. All PCR products were sequenced on an Illumina Hiseq 2500 using 500 cycle version 252 reagent kits