An automated workflow to assess completeness and curate GenBank for environmental DNA metabarcoding: The marine fish assemblage as case study

Abstract

To successfully implement environmental DNA-based (eDNA) diversity monitoring, the completeness and accuracy of reference databases used for taxonomic assignment of eDNA sequences are among the challenges to be tackled. Here, we have developed a workflow that evaluates the current status of GenBank for marine fishes.

Comparison of traditional and molecular surveys of fish biodiversity in southern Te Wāhipounamu/Fiordland (Aotearoa/New Zealand)

“…However, the overall conclusion remains. Of the over 1294 known New Zealand marine fish species, molecular reference data of any kind are available only for 489 species in southern New Zealand, no available primer pairs have sufficient reference data, and the employed 12S marker is among the most popular for fish species assignment (Claver et al., 2023).…”

“…Lacking local reference data, we used a self‐curated copy of the NCBI nucleotide collection (Benson et al., 2011; version September 2022), as an overall aggregate of many 12S sequences, for example, including sequences submitted as part of the Meta‐Fish‐Lib software (Collins et al., 2021), and safeguarding us from misassignments due to a limited search space (Gold et al., 2022). To curate our NCBI‐derived reference data, we excluded 20,008,447 environmental samples, thus minimizing taxonomic misassignments to low‐quality data (Claver et al., 2023). To yield a maximum of taxonomically annotated ASVs, we chose relaxed taxonomic assignment parameters in combination with an e‐value to retain only the most significant alignments.…”

“…For example, for six commonly used 12S primer pairs, recognized as well suited for multispecies fish surveys (Weigand et al, 2019;Zhu & Iwasaki, 2023), an average of 36% of northern European fish species are available as reference data, but only 26% of southern New Zealand species (GAPeDNA v1.0.1 web interface, 11-Sep-2021; Marques et al, 2021; also see Table S1; and reviewed by Marques et al, 2021). Where purposefully generated reference data are unavailable, selected publicly available reference data (such as derived from GenBank) are the only option for taxonomic assignments, and useful if taxonomic assignments are verified in the study context (Balvočiūtė & Huson, 2017;Claver et al, 2023).…”

Increasing marine trophic web knowledge through DNA analyses of fish stomach contents: a step towards an Ecosystem Based Approach to fisheries research

“…Complementary visual inspection of the stomach contents would be still needed to confirm cannibalism and/or whether a prey is more likely to come from direct or indirect consumption. Finally, is should be noted that the accuracy of DNA metabarcoding is highly dependent on the completeness of reference databases (Claver et al, 2023). In this sense, however, metabarcoding will only become a more powerful tool, as reference databases are being continuously populated and increased.…”

Evaluating Metabarcoding Markers for Identifying Zooplankton and Ichthyoplankton Communities to Species in the Salish Sea: Morphological Comparisons and Rare, Threatened or Invasive Species

“…Our research aims to contribute genetic identification methodology, ground-truthed with zooplankton morphological identifications, towards understanding and monitoring the dynamic responses of zooplankton and ichthyoplankton species and their communities in future studies. Here we employ a high-throughput sequence (HTS) metabarcoding assay approach, which prior studies have indicated can capture a broad diversity of taxa that are often difficult to identify using microscopy and/or other morphological techniques; these include many larval bivalves, gastropods, polychaetes, copepods, and fishes [28][29][30][31][32]. Metabarcoding can also detect species that are low in abundance and/or cryptic [3,9,[29][30][31][32][33][34][35][36].…”