eDNA metabarcoding
The sequencing library was generated from PCR amplicons produced using
the primer pair 12S-V5-F (5´-ACTGGGATTAGATACCCC-3´) and 12S-V5-R
(5´-TAGAACAGGCTCCTCTAG-3´), which amplifies a 73-110 bp fragment of the
mitochondrial 12S ribosomal RNA (rRNA) gene in vertebrates (Riaz et al.,
2011), and a ~106 bp fragment in European fishes
(Hänfling et al., 2016). All PCRs were set up using eight-strip PCR
tubes in a UV and bleach sterilized laminar flow hood in the eDNA
laboratory to minimize contamination risk. To detect possible PCR
contamination within each sub-library, we included PCR negative controls
(molecular grade water) and PCR positive controls. The latter used
tissue DNA (0.05 ng/µL) from Zebra mbuna (Maylandia zebra ), a
cichlid from Lake Malawi which is not present in natural waters in the
Czech Republic. The locus primers were attached to 24 unique index
combinations and heterogeneity spacers for the first PCR. The first PCR
for each sample, field/filtration blank, evaporation blank, extraction
blank, PCR negative control and PCR positive control was performed in 25
µL reaction volumes, including 12.5 µL Q5®️High-Fidelity 2X Master Mix (New England Biolabs®Inc., MA, USA), 1.5 µL of each primer (10 µM, Integrated DNA
Technologies), 0.5 µL Thermo Scientific™ Bovine Serum Albumin (Fisher
Scientific, UK), 5 µL DNA, and 4 µL molecular grade water. PCR
conditions were initial denaturation for 5 min at 98 °C, 35 cycles of 10
s at 98 °C, 20 s at 58 °C and 30 s at 72 °C, and final extension for 7
min at 72 °C. Three technical replicates were performed for each sample
and then pooled to minimize PCR stochasticity. Pooled PCR products were
visualized on a 2% agarose gel stained with GelRed (Cambridge
Bioscience, UK). PCR products were normalized into 12 sub-libraries (two
per sampling campaign) based on the band strength observed for each
sample (very bright = 5 µL, bright = 10 µL, faint = 15 µL, and very
faint/no band = 20 µL) together with 10 µL of the filtration blanks,
evaporation blanks, extraction blanks, PCR negative controls and 1 µL of
the PCR positive controls (Alberdi et al., 2018). The normalized
sub-libraries were purified using a double size selection protocol with
Mag-Bind® TotalPure NGS magnetic beads (Omega Bio-tek, USA) to remove
primer dimers and large secondary products (Bronner et al., 2014).
Ratios of 0.9× and 0.15× magnetic beads to 100 μL of each sub-library
were used.
The 12 purified sub-libraries were used as template DNA for a second PCR
attaching Illumina indices. The second PCR was performed in 50 µL
reaction volumes, consisting of 25 µL Q5®️High-Fidelity 2X Master Mix, 3 µL of each primer (10 µM), 4 µL purified
product, and 15 µL molecular grade water. PCR conditions were initial
denaturation for 3 min at 95 °C, 10 cycles of 20 s at 98 °C and 1 min at
72 °C, and final extension for 5 min at 72 °C. PCR was performed for
sub-libraries in duplicate and replicates were pooled before
visualization on a 2% agarose gel stained with GelRed. The
sub-libraries were purified using a double size selection protocol with
magnetic beads to remove secondary products (Bronner et al., 2014).
Ratios of 0.7× and 0.15× magnetic beads to 50 μL of each sub-library
were used. The sub-libraries were normalized and pooled according to the
number of samples each contained (without blanks and PCR negative
controls) and their DNA concentration. The pooled library was purified
again using the same volumes and ratios as the previous purification,
and quantified by qPCR using the NEBNext® Library
Quant Kit for Illumina® (New England
Biolabs® Inc., MA, USA) on a StepOnePlus™ Real-Time
PCR System (Thermo Fisher Scientific, UK) following manufacturer’s
guidelines. Removal of secondary product was verified using an Agilent
2200 TapeStation and High Sensitivity D1000 ScreenTape (Agilent
Technologies, CA, USA). Libraries were run at 13 pM with 10% PhiX
Control v3 on an Illumina MiSeq® using a MiSeq Reagent
Kit v3 (600-cycle) (Illumina, Inc., CA, USA).