DNA extraction, amplification and high-throughput sequencing
The eDNA capsules were processed at SPYGEN using a standard protocol (Polanco Fernández et al. 2021). The DNA extraction, amplification and sequencing were performed in separate dedicated rooms, equipped with positive air pressure, UV treatment and frequent air renewal. Two extractions per filter were performed following the protocol of Pont et al. (2018) and were pooled before the amplification step. After the DNA extraction the samples were tested for inhibition following the protocol described in Biggs et al. (2015). If the sample was considered inhibited, it was diluted 5-fold before the amplification. DNA amplifications were performed in a final volume of 25 μL, using 3 μL of DNA extract as the template using a teleostean primer pairs (teleo/Tele01; Valentini et al. 2016, Taberlet et al. 2018). The amplification mixture contained 1 U of AmpliTaq Gold DNA Polymerase (Applied Biosystems, Foster City, CA), 10 mM Tris-HCl, 50 mM KCl, 2.5 mM MgCl2, 0.2 mM each dNTP, 0.2 μM of each primers, 4 µM human blocking primer for the “teleo” primers and 0.2 µg/µL bovine serum albumin (BSA, Roche Diagnostic, Basel, Switzerland). The tags for the forward and reverse primers were identical. The PCR mixture was denatured at 95°C for 10 min, followed by 50 cycles of 30 s at 95°C, 30 s at 55°C for teleo and finally 1 min at 72 °C. Twelve replicates of PCRs were amplified per filtration. After amplification, the samples were titrated using capillary electrophoresis (QIAxcel; Qiagen GmbH) and purified using the MinElute PCR purification kit (Qiagen GmbH). Before sequencing, purified DNA was titrated again using capillary electrophoresis. We pooled the purified PCR products in equal volumes to achieve a theoretical sequencing depth of 1 000,000 reads per sample. Three libraries were prepared using the MetaFast protocol. The paired-end sequencing (2x125 bp) was carried out on a MiSeq (2x125 bp, Illumina, San Diego, CA, USA) using a MiSeq Flow Cell Kit Version3 (Illumina, San Diego, CA, USA) per each library following the manufacturer’s instructions. Library preparation and sequencing were performed at Fasteris (Geneva, Switzerland). Three negative extraction controls and two negative PCR controls (ultrapure water, 12 replicates) were amplified per primer pair and sequenced in parallel to the samples to monitor possible contaminants.