Maximizing the reliability and the number of species assignments in metabarcoding studies using a curated regional library and a public repository

Abstract

Biodiversity assessments relying on DNA have increased rapidly over the last decade. However, the reliability of taxonomic assignments in metabarcoding studies is variable and affected by the reference databases and the assignment methods used. Species level assignments are usually considered as reliable using regional libraries but unreliable using public repositories. In this study, we aimed to test this assumption for metazoan species detected in the Gulf of St. Lawrence in the Northwest Atlantic. We first created a regional library (GSL-rl) by data mining COI barcode sequences from BOLD, and included a reliability ranking system for species assignments. We then estimated 1) the accuracy and precision of the public repository NCBI-nt for species assignments using sequences from the regional library and 2) compared the detection and reliability of species assignments of a metabarcoding dataset using either NCBI-nt or the regional library and popular assignment methods. With NCBI-nt and sequences from the regional library, the BLAST-LCA (least common ancestor) method was the most precise method for species assignments, but the accuracy was higher with the BLAST-TopHit method (>80% over all taxa, between 70% and 90% amongst taxonomic groups). With the metabarcoding dataset, the reliability of species assignments was greater using GSL-rl compared to NCBI-nt. However, we also observed that the total number of reliable species assignments could be maximized using both GSL-rl and NCBI-nt with different optimized assignment methods. The use of a two-step approach for species assignments, i.e., using a regional library and a public repository, could improve the reliability and the number of detected species in metabarcoding studies.

rCRUX: A rapid and versatile tool for generating metabarcoding reference libraries in R

“…Curation of reference databases is important to ensure accurate taxonomic assignment (Bourret et al., 2023; Fontes et al., 2021). rCRUX performs curation of reference sequences by selecting specific marker genes, filtering sequences to relevant lengths, and collapsing accessions with identical sequences to lowest common ancestor analogous to generating BINs (Fontes et al., 2021).…”

“…However, the curation efforts implemented within rCRUX are limited, and future efforts could implement additional quality assurance and quality control measures. For example, recent efforts have demonstrated that local reference databases often perform better than global ones, thus developing tools to subset reference databases to specific geographic regions of interest would provide a valuable curation step (Blackman et al., 2023; Bourret et al., 2023; Gold et al., 2021). However, implementing such an approach would require accurate global repositories of species distributions and detailed inventories of regional biodiversity which are often not readily available (Beck et al., 2014; Costello & Berghe, 2006).…”

“…Curation of reference databases is important to ensure accurate taxonomic assignment (Bourret et al, 2023;Fontes et al, 2021).…”

A comprehensive evaluation of taxonomic classifiers in marine vertebrate eDNA studies

“…Custom curated reference databases have been shown to increase reliability and species-level detections in eDNA studies (Collins et al, 2021, Bourret et al, 2023, Jeunen et al, 2023). We built gene-specific reference databases for 12S rDNA, 16S rDNA, and COI including marine vertebrates that inhabit the Australian Exclusive Economic Zone (EEZ).…”

“…A notable scarcity of eDNA-based benchmarks for marine vertebrates exists (Mathon et al, 2021), with few studies comparing taxonomic classifiers against known data. Bourret et al (2023) tested different BLAST parameters using a regional curated subset of Metazoans of the BOLD database, revealing that database curation increased the number of species detected. To our knowledge, Mathon et al (2021) conducted the sole marine vertebrate study available to date using simulated communities where they compared 13 bioinformatic programs and pipelines and recommended VSEARCH for taxonomic assignments.…”