This SOP describes the protocol used for metabarcoding environmental sea water and sediment samples employing Illumina NGS technology. Although the primers deployed here are described to cover a broad range of fish species (Miya et al., 2015), it remains to be evaluated, if all relevant North Sea species can be detected. However, different primer sets can be utilized to address bony and cartilaginous fishes. Furthermore, public databases such as NCBI and ENA are far from being complete and do not contain 12S rDNA gene sequences for quite a few common fish species, which makes the detection of these species impossible or leads to incorrect records of closely related species. In the presence of a high background noise of non-fish DNA (as is always the case in eDNA samples) the Mi-Fish primers amplify other sequences (mostly microorganisms) as well. It is therefore recommended to perform all PCRs with the KAPA polymerase, which is working reliably at higher annealing temperatures (65 °C). The poly(N) bases at the 5’ ends of the tag primers are important to ensure the specificity to fish. It is further strongly recommended to perform purification steps after the 1st and the 2nd PCR. Particularly the isolation and purification of the correctly sized band is necessary to obtain predominantly fish sequence reads in the subsequent NGS analyses. A separation of the 2nd PCR product on a 2% agarose gel is necessary.
Hanel et al. (Tue,) studied this question.