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).