title: 16S rRNA high-throughput sequencing of loach gut: under chronic ammonia nitrogen stress
design: DNA extraction and PCR amplification were performed as follows: genomic DNA was extracted from each sample using the Qubit 2.0 DNA Detection Kit (Life, Q10212) according to the manufacturer's instructions. The quality of the extracted DNA was assessed using 1.0% agarose gel electrophoresis and spectrophotometry, and the optical density was measured at a 260 nm/280 nm ratio. All extracted DNA samples were then stored at -20 C for subsequent analysis. To investigate the structure and composition of the bacterial communities in the loach gut, high-throughput sequencing of the 16S rRNA gene was conducted. The V3V4 variable region of the bacterial 16S rRNA gene was amplified using the universal forward 338/806 primers (338F: ACTCCTACGGGAGGCAGCCA and 806R GGACTACVSGGGTATCTAAT) primers. Raw data were screened using Quantitative Insights into Microbial Ecology (QIIME) based on barcode and primer sequences.