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.