Bacteria are everywhere, even in your COI data: Τhe art of getting to know the unknown unknowns and shine light on the dark matter!

Environmental DNA (eDNA) metabarcoding has been commonly used in recent years (Jeunen et al. 2019) for the identification of species composition environmental samples. By making use genetic markers anchored conserved gene regions, universally present acrooss large taxonomy groups, eDNA exploits both extra- and intra-cellular fragments biodiversity assessment. However, there is not a truly “universal” marker that capable amplifying all across different taxa (Kress 2015). The mitochondrial cytochrome C oxidase subunit I (COI) many desirable properties “universal" widely assessing identity Eukaryotes, especially metazoans (Andjar 2018). great number COI Operational Taxonomic Units (OTUs) or/and Amplicon Sequence Variants (ASVs) retrieved from such studies do match reference sequences are often referred to as “dark matter” (Deagle 2014). aim this study was discover origins identities these dark matter sequences. We built phylogenetic tree included COI-sequence-related information life possible. An overview steps followed presented Fig. 1a. Briefly, Midori 2 database retrieve eukaryotes (183,330 species). In addition, API BOLD source corresponding Bacteria (559 genera) Archaea (41 Consensus at family level were constructed each three initial datasets. COI-oriented then by using 1,240 consensus with more than 80% those coming eukaryotic taxa. Phylogeny-based taxonomic assignment place query a) total sequences, b) assigned Eukaryotes c) unassigned subsets OTUs, marine freshwater samples, during in-house experiments, placed (Fig. 1b). It clear proportion targeting region actually represents bacterial branches conclude may come bias derived amplification sequencing taxa, depending on primer pair used. time being, publicly available far too few represent variability; thus, reliable them suggest should be databases OTUs/ASVs COI-based eukaryote allow amplified excluded enabling researchers exclude non-target Further, approach here allows better understand unknown unknowns shed light their sequence data.