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

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
relies 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. Here, we reviewed the literature and compiled a
list of 22 primer sets for eDNA-based 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. 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.