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.