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.