Bioinformatics
After obtaining valid sequences from all samples, the quality of the sequence fragments was evaluated, and fragments < 100 bp in length were discarded. Based on their overlapping relationships, paired fragments were spliced into sequences. Finally, high-quality sequences for each sample were obtained by splitting them based on the barcode and primer sequences, with the sequence direction corrected according to the positive and negative barcodes and primer orientations.
Operational taxonomic unit (OTU) clustering was performed using MitoFish (http://mitofish.aori.u-tokyo.ac.jp/) and NCBI (https://www.ncbi.nlm.nih.gov/) databases as data sources. To create a custom database, all fish data from the Chishui River were retrieved and downloaded. To ensure data integrity, the scope of the searches was expanded for comparison with the entire NCBI database. After comparison and annotation, the corresponding OTU and species abundance tables were obtained.