DNA amplification
The DNA was amplified using via polymerase chain reaction (PCR). For the 12S rRNA gene, NeoFish (Milan et al., 2020) and MiFish (Miya et al., 2015) markers were amplified, and the fragment of the COI gene was amplified using a cocktail of primers targeting the standard COI fragment (Ward et al., 2005). Each sample was amplified in triplicate using the primer with a specific barcode tag for demultiplexing. The PCR reaction solution had a final volume of 20µl, containing: 8.34µl of ultrapure water, 0.16µl of BSA (100µg/ml), 10µl of AmpliTaq™ Gold 360 Master Mix (Thermo Fisher), 0.25µl of each primer, and 1,0µl of DNA template. An additional 1,0µl of ultrapure water was added for the negative control samples instead of the DNA template. For the positive control sample, 1.0µl of template DNA from a saltwater fish species,Prionace glauca , was added.
PCR conditions consisted of initial denaturation for 10 min at 95°C, followed by 35 cycles of denaturation for 1 min at 95°C, primers annealing for 30 sec at 56°C (COI), 60°C (MiFish) or 63°C (NeoFish) and extension for 30 sec at 72°C, with a final extension for 7 min at 72°C.
The PCR results were checked using 1.8% agarose gel electrophoresis. Both 12S markers presented bands of the expected size for all samples, including the positive control, whereas for COI, the sample SF08 failed to produce any bands but was also included in the sequencing step. The negative control samples did not produce any bands but were also sequenced with the other samples.