The Dark mAtteR iNvestigator (DARN) tool: getting to know the known unknowns in COI amplicon data

The mitochondrial cytochrome C oxidase subunit I gene (COI) is commonly used in environmental DNA (eDNA) metabarcoding studies, especially for assessing metazoan diversity. Yet, a great number of COI operational taxonomic units (OTUs) or/and amplicon sequence variants (ASVs) retrieved from such studies do not get assignment with reference sequence. To assess and investigate sequences, we have developed the Dark mAtteR iNvestigator (DARN) software tool. For this purpose, COI-oriented phylogenetic tree was built 1,593 consensus sequences covering all three domains life. With respect to eukaryotes, at family level were constructed 183,330 Midori 2 database, which represented 70% initial sequences. Similarly, 431 bacterial 15 archaeal taxa (29% 1% respectively) BOLD PFam databases. DARN makes use pre-processed samples provide both tabular graphical overview their assignments. evaluate DARN, bulk different aquatic environments using various primer sets analysed. We demonstrate that large proportion non-target prokaryotic organisms, as bacteria archaea, are also amplified eDNA suggest be included databases taxonomy allow further analyses dark matter. source code available on GitHub https://github.com/hariszaf/darn Docker image https://hub.docker.com/r/hariszaf/darn.