4 DISCUSSION
Metabarcoding-based biomonitoring requires the availability of primers with high performance, as they must amplify all the relevant target taxa, have sufficient resolution to identify them at the desired taxonomic level, and amplify short sequences usable with eDNA (Ficetolaet al. 2010; Taberlet et al. 2018). Finding primers with all these features is challenging, and the identification of ”perfect” metabarcodes has often been labelled as a ”search for the Holy Grail” (Rubinoff et al. 2006). By combining an extensive in vitroanalysis with the assessment of publicly-available sequences, our study highlights the complexity of finding all these desired features in one single metabarcode. It also provides a comparison of performances, allowing the identification of most appropriate primers for different aims and taxonomic groups, and it produced a reference database for the taxonomic identification of a large number of benthic insects.