DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work

Abstract

100Effective identification of species using short DNA fragments (DNA barcoding and DNA 101 metabarcoding) requires reliable sequence reference libraries of known taxa. Both 102 taxonomically comprehensive coverage and content quality are important for sufficient 103 accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are 104 particularly important if molecular identification tools are to be implemented in biomonitoring 105 and reports in the context of the EU Water Framework Directive (WFD) and the Marine 106Strategy Framework Directive (MSFD). We analysed gaps in the two most important 107 reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a 108 focus on the taxa most frequently used in WFD and MSFD. Our analyses show that 109 coverage varies strongly among taxonomic groups, and among geographic regions. In 110 general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, 111 caddisflies and vascular plants) are well represented in the barcode libraries, while others 112 have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also 113 found that species monitored in several countries often are represented by barcodes in 114 reference libraries, while species monitored in a single country frequently lack sequence 115 records. A large proportion of species (up to 50%) in several taxonomic groups are only 116represented by private data in BOLD. Our results have implications for the future strategy to 117 fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the 118 monitoring of European aquatic biota under the WFD and MSFD. For example, missing 119 species relevant to monitoring in multiple countries should be prioritized. We also discuss 120 why a strategy for quality control and quality assurance of barcode reference libraries is 121 needed and recommend future steps to ensure full utilization of metabarcoding in aquatic 122 biomonitoring. 123 124

Uncovering the complete biodiversity structure in spatial networks: the example of riverine systems

“…They are, however not universal for all organisms, and currently the taxonomic assignment is mostly restricted by the lack of complete and adequate reference databases. Important ways forward are thus: 1) the design or optimisation of primers (both their specificity but also generality) (Elbrecht and Leese 2016, Macher et al ), 2) to complement and fill the respective databases (Blackman et al , Weigand et al ), and 3) to possibly think of whole‐mitochondrial sequencing based on eDNA samples (Deiner et al ), in order to combine data of multiple markers from the same organism. All three areas are under ongoing research and major progress is being made.…”

Reference databases, primer choice, and assay sensitivity for environmental metabarcoding: Lessons learnt from a re‐evaluation of an eDNA fish assessment in the Volga headwaters

“…After data generation and raw data filtration, the retrieved DNA sequences are usually compared with a reference database of the expected species community in order to translate the obtained molecular operational taxonomic units into biological species for final data interpretation. However, such reference databases are still far from complete despite global initiatives like the international Barcode of Life (iBOL; Weigand et al, ). Additionally, the target fragment of iBOL, a part of the mitochondrial cytochrome oxidase subunit I (COI), has been shown to be less suitable for metabarcoding work for vertebrates (Deagle, Jarman, Coissac, Pompanon, & Taberlet, ).…”

“…Therefore, other mitochondrial markers, particularly fragments of the mitochondrial 12S and 16S rRNA genes, have been the focus for metabarcoding primer design for this group (Evans et al, ; Miya et al, ). Databases for these gene fragments are less complete than for COI and have been shown to be heavily biased for certain geographic regions and taxonomic groups (Belle, Stoeckle, & Geist, ; Weigand et al, ), highlighting the need to expand databases for understudied areas and taxonomic groups.…”

Identifying error and accurately interpreting environmental DNA metabarcoding results: A case study to detect vertebrates at arid zone waterholes

“…Incomplete databases can also lead to a sequence read being incorrectly assigned to a single species with perfect (or near perfect) sequence identity and, if no related taxa are present in the database, the incorrect assignment may go unnoticed (Curry, Gibson, Shokralla, Hajibabaei, & Baird, 2018; Geiger et al., 2016; Weigand et al., 2019). Utilizing an alternative or additional gene region may provide improved representation or resolution for particular taxonomic groups and reduce misassignment error (Deagle, Jarman, Coissac, Pompanon, & Tab erlet, 2014; Tang et al., 2014).…”