DNA extraction and sequencing
A DNeasy Blood & Tissue Kit (Qiagen, Düsseldorf, Germany) was used to extract DNA. Given the low content and concentration of fish DNA in the water, the number of elution cycles was increased from two to four to enhance the concentration of the extracted DNA according to the manufacturer’s instructions. To determine the amount of DNA to be added to the PCR, a Qubit3.0 DNA detection kit (Invitrogen, California, USA) was used to accurately quantify genomic DNA. The MiFish PCR primers were as follows: forward: 5′-GTCGGTAAAACTCGTGCCAGC-3′ and reverse: 5′-CATAGTGGGGTATCTAATCCCAGTTTG-3′. Each PCR reaction consisted of 15 mL 2× Hieff® Robust PCR Master Mix (Yeasen, Shanghai, China), 1 μL forward Bar-PCR primer, 1 μL reverse Primer, 10–20 ng PCR product, Illumina bridge PCR-compatible primers, and 9–12 μL H2O, for a total volume of 30 μL. The PCR reaction conditions were as follows: 94 °C for 3 min; 5 cycles at 94 °C for 30 s, 45 °C for 20 s, and 65 °C for 30 s; 27 cycles at 94 °C for 20 s, 55 °C for 20 s, and 72 °C for 30 s; 72 °C for 5 min; and then maintained at 10 °C. A PCR negative control was prepared using ddH2O as the template to assess for potential contamination. Three replicates were performed for each sample during PCR amplification and the PCR products were mixed. PCR products were detected by 2% agarose gel electrophoresis. Shenggong Bioengineering Co., Ltd. (Shanghai, China) spliced the PCR products, and high-throughput sequencing was conducted using the Illumina NovaSeq 6000 (Illumina, California, USA) sequencing platform.