Disentangling the components of coastal fish biodiversity in southern Brittany by applying an environmental DNA (eDNA) approach.

Data acquisition During three days, from September 8th to September 10th 2020, we sampled 17 stations whose 13 - located near to the coast - as transects and 4 - located deeper - as fixed points. For each station, we performed two water filtration replicates in parallel on each side of the boatcorresponding to a total of 34 filters for the whole area. We followed a precise protocol to sample eDNA based on material composed of an Athena® peristaltic pump, a VigiDNA® 0.22 µM cross‐flow filtration capsule making it possible to filter a large water volume of 30L and disposable sterile tubing for each filtration capsule. At the end of each filtration, the water inside the capsules was emptied and filled with 80 ml of CL1 conservation buffer and stored at room temperature. To avoid any contamination, we carried out all the sampling steps using disposable gloves and single-use filtration equipment. edNA extraction, amplification and sequencing DNA extraction, amplification and high-throughput sequencing were performed in distinct dedicated rooms set up with positive air pressure, UV treatment and frequent air renewal. After the DNA extraction, we tested the samples 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. 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 and 0.2 µg/µL bovine serum albumin (BSA, Roche Diagnostic, Basel, Switzerland). To perform the amplification we used the teleo primer (forward: ACACCGCCCGTCACTCT, reverse: CTTCCGGTACACTTACCATG) that amplify a region of c.a. 60 bases pair of mitochondrial 12S region. This primer pair was designed to capture teleosteans taxa (Valentini et al., 2016) but also capture Elasmobranchii taxa (Polanco-Fernández et al., 2021b). The “teleo” primers were 5’-labeled with an eight-nucleotide tag unique to each PCR replicate (with at least three differences between any pair of tags), allowing the assignment of each sequence to the corresponding sample during sequence analysis. The tags for the forward and reverse primers were identical for each PCR replicate. 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 and 1 min at 72 °C and a final elongation step at 72°C for 7 min. Twelve replicates of PCRs were run per filtration (i.e., 24 per sampling site). 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. Library preparation and sequencing were performed at Fasteris (Geneva, Switzerland). Two libraries were prepared using the MetaFast protocol and the paired-end sequencing (2x125 bp) was carried out using on a MiSeq (2x125 bp, Illumina, San Diego, CA, USA) and the MiSeq Flow Cell Kit Version3 (Illumina, San Diego, CA, USA) were used following the manufacturer’s instructions. Two negative extraction controls and one negative PCR control (ultrapure water) were amplified (12 replicates as well) and sequenced in parallel to the samples to monitor possible contaminants.