title: Microbiota of Nile tilapia: Intestine
design: Individual intestines from three fish in each treatment were aseptically dissected and the intestinal contents were used for bacterial composition analysis. Total bacterial DNA was isolated using a E.Z.N.A. Soil DNA Kit (OMEGA, USA) according to the manufacturer s instructions. DNA quantity and quality were measured using a NanoDrop 2000 Spectrophotometer (Thermo, USA). The V3-V4 regions of the bacteria 16S ribosomal RNA genes were amplified by polymerase chain reaction (PCR) using primers 338F: 5 -ACTCCTACGGGAGGCAGCA-3  and 806R: 5 -GGCTACHVGGGTWTCTAAT-3 . Unique eight-base barcodes were added to each primer to distinguish PCR products. PCR reactions were performed in 20  L mixture containing 4  L of 5   Fast Pfu Buffer, 2  L of 2.5 mM dNTPs, 0.8  L of each primer (5  M), 0.4  L of Fast Pfu Polymerase (Trans Gen, China), and 10 ng of template DNA. The PCR conditions were as follows: 95  C for 3 min for DNA denaturation, with the amplification lasting 29 cycles (95  C for 30 s, 55  C for 30 s and 72  C for 45 s), and final extension for 10 min at 72  C. Purified PCR products were subjected to high-throughput sequencing using Illumina HiSeq 2500 (Majorbio Bio-Pharm Technology, Co., Ltd., Shanghai, China). The raw pair-end readings obtained were subjected to quality-control procedures by using the quantitative insights into microbial ecology (QIIME, version 1.17). The qualified reads were clustered to generate operational taxonomic units (OTUs) at the 97% similarity level by using UPARSE (version 7.1). Chimeric sequences were identified and removed using UCHIME. The phylogenetic affiliation of each 16S rRNA gene sequence was analyzed by RDP Classifier against the silva 16S rRNA database using confidence threshold of 70%. Taxonomic richness and diversity estimators were determined using a Mothur software.