2.3 Environmental DNA extraction and metabarcoding
Within 24 hr of collection, all water samples were filtered using Whatman glass microfiber filter papers (47 mm diameter, 1.2 μm pore size). Prior to filtering each river sample, 500 ml of ddH2O was filtered on a separate filter to act as laboratory controls, followed by filtration of the river sample on new filters using the same filtration apparatus. DNA from water was extracted using the Qiagen DNeasy Tissue and Blood DNA extraction kit following the manufacturer’s protocol with minor modifications. Three membranes (500 mL of water per membrane) for each sample were cut into pieces, ground and mixed. Then, the sample was soaked in 600 µL of 2 × lysis buffer and 40 µL of proteinase K. Incubation with this mixture was performed at 56 °C for 2.5 hr. Finally, we washed the filters in the mixture and performed elution in 200 µL of AE buffer. Filtration blanks and negative controls were coextracted alongside the samples and were subjected to the same protocol as the samples. The DNA concentration was determined using the Qubit dsDNA HS Assay Kit and detected in a 1.0% agarose gel. No data or bands were observed for the filtration blanks or negative controls.
Metabarcoding was performed in duplicate on each DNA extract with the primers MiFish-F (5’GTCGGTAAAACTCGTGCCAGC-3’) and MiFish-R (5’-CATAGTGGGGTATCTAATCCCAGTTTG-3’) (Miya et al., 2015), which target the 12S rDNA region (amplifying an ~180 bp region) of the mitochondrial genome, to identify fish species. DNA amplifications were performed in a two-step PCR protocol designed for the BGISEQ-500 platform. Three PCR replicates were performed for each sample. For each set of replications, environmental samples, filtration blanks and negative controls were included. The PCR assay volume was 50 µL, including 0.3 µL of Takara Ex Taq (5 U/µL), 5 µL of 10×Ex Taq buffer (20 mM Mg2+ plus), 4 µL of a dNTP mixture, 1 µL of the forwards and reverse primers (10 µM), 1 µL of the DNA template (environmental samples, filtration blanks and negative controls), and molecular biology-grade water added to 50 µL. For all samples, the first-step PCR was performed as follows: 94 °C for 5 min, followed by 10 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, with a final extension at 72 °C for 10 min. The first-step PCR products were diluted 5 times with molecular biology grade water and used as the templates for the second-step PCR. For subsequent sequencing on the BGISEQ-500 platform, two or three random nucleotides were inserted into the MiFish primers (to increase sequence diversity during sequencing). The second-step PCR was carried out in a 50 µL reaction volume including 0.3 µL of Takara Ex Taq (5 U/µL), 5 µL of 10× Ex Taq buffer (20 mM Mg2+ plus), 4 µL of a dNTP Mixture, 1 µL of the forwards and reverse primers with the BGISEQ-500 adapter (10 µM), 2 µL of the template, and molecular biology-grade water added to 50 µL. The PCR procedure was as follows: 94 °C for 5 min, followed by 20 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s, with a final extension at 72 °C for 10 min. After the two-step PCR amplification, the PCR products were detected in a 1.5% agarose gel. None of the filtration blanks or negative controls showed amplification.