2.3 DNA amplification
Two primer pairs were used, each amplifying either the Unionida or the
Venerida (Prié et al., 2020; presented in Table 1). Each DNA extract was
amplified in twelve replicates per primer pair, including all the
extraction negative controls and the PCR negative controls.
The amplification mixture contained 1 U of AmpliTaq Gold Polymerase
(Applied Biosystems), 1x PCR Gold buffer containing 2 mM of MgCl2, 0.2
mM of each dNTPs, 0.5 μM of each tagged forward and reverse primers and
0.2 mg/mL of bovine serum albumin (BSA, Roche Diagnostics). The final
volume was 25 μL including 3 μL of eDNA extraction. All amplification
underwent the same PCR parameters (initial denaturation for 3 min at
95°C, 50 cycles of 30 s at 95°C, 30 s at 50°C and 30 s at 72°C, and
final elongation at 72°C for 5 min). Forward and reverse primers were
5‘-labeled with identical eight nucleotides tags to enable the
subsequent assignment of sequences to their respective sample. Each tag
had at least four differences with other tags to properly assign reads
to samples. A total of 65 negative extraction controls and 35 PCR
negatives controls (ultrapure water) were also included in the
experiment to monitor possible contaminations. Pooled PCR products were
purified using a MinElute PCR purification kit (Qiagen), with elution in
15 μl buffer.