Fish mock community for eDNA metabarcoding
Environmental DNA (eDNA) metabarcoding has become a powerful tool for examining fish communities. The demand for methodological standardization and the implementation of eDNA-based assessments into the regulatory monitoring (e.g., Water Framework Directive) are imminent. To ensure methodical accuracy and to meet regulatory standards, various sampling, laboratory and bioinformatic workflows have been established. However, a crucial prerequisite for a comprehensive fish monitoring is the choice of suitable primer pairs to accurately depict the present fish fauna. Various fish-specific primer pairs targeting different genetic marker regions were published over the past decade. However, a dedicated study to evaluate performance of frequently applied fish primer pairs to assess Central European fish species has not yet been conducted. Therefore, we created an artificial community composed of DNA from 45 Central European fish species and examined the discriminatory power and reproducibility of five fish primer pairs. Our study highlights the effect of the primer choice and bioinformatic filtering on the outcome of eDNA metabarcoding results. From the five primer pairs evaluated in our study the tele02 (12S gene) primer pair proved to be best choice for eDNA metabarcoding of Central European freshwater fish. Here, the MiFish-U (12S) and SeaDNA-mid (COI) primer pairs also displayed good discriminatory power and reproducibility. However, more general primer pairs (i.e., targeting vertebrates) were found to be less reliable and generated high numbers of false-positive and false-negative detections. Our study illustrates how careful selection of primer pairs and bioinformatic pipelines can make eDNA metabarcoding a more reliable tool for fish monitoring.