Sample set
The three assays were tested on seven typologies of samples, including both tissue/biological residues DNA (n=3) and marine eDNA (n=4) templates, encompassing three kinds of positive controls, two negative controls and two trials on Mediterranean marine eDNA “opportunistic” samples already available from ongoing projects from EV’s research group (Table 1). The seven categories are described in details in Box 1.
The DNA from the tissue sample (category 1) was extracted using the Qiagen DNeasy Blood and Tissue Kit, while all remaining DNA samples (categories 2-6) were extracted using the Qiagen DNeasy PowerSoil Kit according to the manufacturer protocol. When biological residual samples (MmoR, category 2) were in liquid form (e.g. feces dissolved in ethanol), an aliquot was filtered through a membrane that was subsequently rinsed with distilled water prior DNA extraction with the same extraction-protocol used for eDNA samples. Table S1 shows a detailed description of all 73 samples.
The four sets of eDNA samples (categories 3 to 5 and 7), coming from different projects and having been collected in different contexts and by different operators, were not always homogeneous in terms of number of liters of marine water being processed and porosity of filters used. However, they were all collected from the sea surface, with the exception of ferry samples collected from sea water intake placed 4.5m below sea level (Valsecchi et al ., in prep.). Water volumes were collected and stored in the Bag-in-Box Sampling System (BiBSS, Valsecchiet al . in prep.).
The two samples collected directly from shore (MmoMc11 and MnoMc12, category 4) underwent through a 10-fold template dilution prior amplification, a procedure used to minimize the interference of PCR inhibitors (Gasparini et al . 2020), known to be particular abundant in proximity of sediments (Lance & Guan 2019).