eDNA capture and extraction
All samples were filtered within 24 hours of collection in a
decontaminated laboratory (bleached floors and surfaces) that is not
used for handling fish specimens or DNA. We filtered 1 L of each sample
(2 x 0.5 L) through two sterile mixed cellulose acetate and cellulose
nitrate filters (47 mm diameter, 0.45 µm porosity, Whatman, UK) using
Nalgene filtration units in combination with a vacuum pump. The filters
were folded using sterile tweezers, placed in 1.5 mL Eppendorf tubes
filled with 96% ethanol, and stored in the dark at 4 °C. Filtration
equipment was sterilized in 10% commercial bleach solution for 10 min,
followed by soaking for 10 min in 10% microsol solution, and rinsed
with purified water after each filtration round. Field blanks also
served as filtration blanks, that were run before the first filtration
round, during filtration, and after the last filtration round to monitor
potential contamination.
In March 2019, the filters in ethanol were transported at room
temperature (six days) to a dedicated eDNA facility at the University of
Hull. In a UV and bleach sterilized laminar flow hood, filters were
removed from ethanol onto individual sterile Petri dishes to dry for
approximately 30 min. Dry filters from the same sample were transferred
to a 5 mL screw cap tube (Axygen™, Fisher Scientific, UK). The storage
ethanol was transferred to a sterile 1.5 mL Eppendorf tube and
centrifuged for 40 min at 11,000 × g. The supernatant was discarded and
the dry pellet dissolved in 50 µL molecular grade water. The eluate was
added to the appropriate 5 mL screw cap tube containing dry filters. For
each sampling campaign, two sterile filters washed with 99% ethanol
were processed alongside sample filters to act as evaporation blanks and
monitor possible contamination. All dried filters were stored at -20 °C
for one week until DNA extraction.
DNA was extracted following the Mu-DNA water protocol (Sellers et al.,
2018). A subset of samples were initially extracted to test extraction
protocol efficacy. Based on relatively low DNA concentrations (average
7.7 ng/µL) measured by NanoDrop 1000 Spectrophotometer, the original
protocol was modified to increase DNA yield. The volumes of lysis
solution and water lysis additive were increased to 900 µL and 300 µL
respectively. To enhance lysate yield, the centrifugation step was
extended to 2 min, and all lysate was transferred to a sterile tube with
flocculant solution for the inhibitor removal step. Finally, the eluate
was transferred back onto the spin column membrane, incubated at room
temperature, and centrifuged to increase DNA yield. To minimize
contamination risk between reservoirs, DNA extraction was performed
separately for each sampling campaign. DNA was extracted from all
filters, field/filtration blanks, evaporation blanks, and extraction
blanks (i.e. extraction buffers only) for each sampling campaign. An
extraction blank was included for each batch of extractions (two per
sampling campaign), that were subsequently pooled to provide one
extraction blank for each sampling campaign. Extracted DNA was stored at
-20 °C for one week until PCR amplification.