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.