A comprehensive and comparative evaluation of primers for metabarcoding eDNA from fish

Abstract

1. Accurate assessments of fish species diversity and community composition are essential for understanding fish ecology and conservation management. Environmental DNA (eDNA) metabarcoding has become an integrated method for monitoring fish species. The accuracy and efficacy of eDNA metabarcoding rely heavily on the choice of primers used for PCR amplification. A wide selection of metabarcoding primers for fish has been developed; however, there exists no comprehensive and comparative evaluation of their amplification or taxonomic classification of a rich diversity of fish species, which hinders informed decisions regarding their suitability for different study systems. 2. Here we reviewed the literature and compiled a list of 22 primer sets for eDNAbased metabarcoding analysis of teleost fish, the performance of which was compared using in silico PCR, followed by in vitro metabarcoding analysis using eDNA from waterbodies in Beijing, which harbour a high number of freshwater fish species. 3. We found that the primers showed considerable differences in the amplified taxonomic ranges and proportions, fish taxa richness, species discrimination power and fish community compositions, both in silico and in vitro. The number of fish taxa detected from eDNA by the primer sets varied from 0 to 66. Primers targeting the 12S rRNA gene generally detected greater fish diversity than those targeting the 16S rRNA or COI genes, while primers targeting the cytochrome b gene amplified the fewest fish taxa in vitro. 4. Regarding target genes, 12S primers generally outperformed other primers in terms of amplified fish diversity. The results of in silico PCR and in vitro tests were not always in agreement, suggesting that primer choice for biodiversity surveys should not be based solely on in silico evaluation. The use of different primers can qualitatively and quantitatively affect the detected biodiversity and these effects should be considered in experimental design and data interpretation. These results will assist with primer selection for eDNA-based fish surveys, and consequently support conservation of freshwater biodiversity.

Computational Evaluation of DNA Metabarcoding for Universal Diagnostics of Invasive Insect Pests

“…Despite this study being a purely in-silico evaluation and the many limitations that this entails (Alberdi et al, 2018; Corse et al, 2019; Zhang et al, 2020), it represents a comprehensive first step in applying big data principles to inform development of HTS based diagnostics for invasive insect pests. As well as providing a starting point for diagnosticians and researchers selecting mini-barcode primers for insect identification, our results offer a degree of confidence to managers and regulators grappling with the consequences of non-targeted HTS assays and how to appropriately respond the incidental detection of regulated species (Darling et al, 2020).…”

Environmental DNA for biomonitoring

“…We focus on fish because they are among the most important groups of bioindicators and also because their study from an eDNA perspective is the most advanced (Pont et al, 2021 ). The barcoding reference database of common fish species in some regions is close to completeness (Knebelsberger et al, 2015 ), fish‐specific markers are well defined (M. Miya et al, 2015 ; Valentini et al, 2016 ; Zhang et al, 2020 ) and protocols for fish eDNA sampling and processing are well established (Masaki Miya et al, 2020 ; Valentini et al, 2016 ). Currently, considerable efforts are directed to solve the most challenging issue, which is related to quantitative fish eDNA data and its application for inferring fish indices in routine biomonitoring.…”

Environmental DNA for biomonitoring

“…We focus on fish because they are among the most important groups of bioindicators and also because their study from an eDNA perspective is the most advanced (Pont et al, 2021). The barcoding reference database of common fish species in some regions is close to completeness (Knebelsberger, Dunz, Neumann, & Geiger, 2015), fish-specific markers are well defined (M. Miya et al, 2015;Valentini et al, 2016;Zhang, Zhao, & Yao, 2020) and protocols for fish eDNA sampling and processing are well established (Masaki Miya, Gotoh, & Sado, 2020;Valentini et al, 2016). Currently, considerable efforts are directed to solve the most challenging issue, which is related to quantitative fish eDNA data and its application for inferring fish indices in routine biomonitoring.…”