A large fraction of ASVs does not receive taxonomic assignment, despite the well-known species composition in the samples
Previous COI metabarcode datasets derived from macrobenthos communities have shown that only a small fraction of OTUs receives taxonomic assignment linked to macrobenthos (Aylagas et al., 2018; Leray & Knowlton, 2015) which has been explained by the incompleteness of reference databases (Leray & Knowlton, 2015; Wangensteen, Palacin, Guardiola, & Turon, 2018). Our datasets also contained a low percentage of ASVs (4 - 22.6%) with taxonomic assignment. Even when using the MIDORI reference database with almost 47 000 marine metazoan species for taxonomic assignment, only an additional 0.6-8.6% of the ASVs were assigned. Previous macrobenthos metabarcode studies reported a high portion of non-metazoan taxonomic assignments (Aylagas et al., 2018; Wangensteen et al., 2018). We tested this for primer set A, by blasting all ASVs that were unassigned after Midori (1471 ASVs) against the nt database and found only 45 ASVs with a non-metazoan hit. It remains unclear whether the remaining unassigned ASVs represent unknown diversity, genetic noise e.g. created by PCR and/or sequencing errors, nuclear pseudogenes, or aspecific amplification of other genomic regions.