In silico and empirical evaluation of twelve metabarcoding primer sets for insectivorous diet analyses

Abstract

During the most recent decade, environmental DNA metabarcoding approaches have been both developed and improved to minimize the biological and technical biases in these protocols. However, challenges remain, notably those relating to primer design. In the current study, we comprehensively assessed the performance of ten COI and two 16S primer pairs for eDNA metabarcoding, including novel and previously published primers. We used a combined approach of in silico, in vivo‐mock community (33 arthropod taxa from 16 orders), and guano‐based analyses to identify primer sets that would maximize arthropod detection and taxonomic identification, successfully identify the predator (bat) species, and minimize the time and financial costs of the experiment. We focused on two insectivorous bat species that live together in mixed colonies: the greater horseshoe bat (Rhinolophus ferrumequinum) and Geoffroy's bat (Myotis emarginatus). We found that primer degeneracy is the main factor that influences arthropod detection in silico and mock community analyses, while amplicon length is critical for the detection of arthropods from degraded DNA samples. Our guano‐based results highlight the importance of detecting and identifying both predator and prey, as guano samples can be contaminated by other insectivorous species. Moreover, we demonstrate that amplifying bat DNA does not reduce the primers' capacity to detect arthropods. We therefore recommend the simultaneous identification of predator and prey. Finally, our results suggest that up to one‐third of prey occurrences may be unreliable and are probably not of primary interest in diet studies, which may decrease the relevance of combining several primer sets instead of using a single efficient one. In conclusion, this study provides a pragmatic framework for eDNA primer selection with respect to scientific and methodological constraints.

eDNA metabarcoding reveals a core and secondary diets of the greater horseshoe bat with strong spatio‐temporal plasticity

“…(2017) (fwhF1: 5′ YTCHACWAAYCAYAARGAYATYGG 3′; fwhR1: 5′ ARTCARTTWCCRAAHCCHCC 3′). These primers offer the best compromise to (a) maximize the detection and identification of arthropod prey and (b) identified the bat species to separate R. ferrumequinum and M. emarginatus diets (Tournayre et al., 2020). We used the metabarcoding protocol described in Galan et al.…”

“…(2018) with the PCR programs optimized in Tournayre et al. (2020). We included a negative control for extraction (NC ext ), a negative control for PCR (NC PCR ), and a negative control for indexing (Nc index ) in each 96‐well microplate.…”

“…Lastly, we considered that a sample was positive for a particular OTU if all replicates (3 out of 3) were positive for this taxon. This procedure enabled the removal of inconsistent OTUs due to PCR or sequencing errors, and reduced the number of putative false‐positive results (Alberdi et al., 2018; Tournayre et al., 2020). The expected mean number of reads after merging the three technical PCR replicates is around 20,000 reads per sample.…”

Primer biases in the molecular assessment of diet in multiple insectivorous mammals

“…flying or semi-aquatic). While it has been acknowledged that the best primer combination for detecting invertebrate prey in one system may not be the best for another (Tournayre et al 2020), there has been a lack of studies investigating this directly. It is therefore important to directly compare the effect of various primers on multiple insectivores occupying different ecological systems (Corse et al 2019).…”

“…Applying these measures can result in the detection of hundreds of species in a bat's diet without losing information to host co-amplification (although it is worth noting that host co-amplification can benefit a bat dietary study by simultaneously detecting a wide range of prey taxa and confirming the predator species from faecal samples; Galan et al 2018;Tournayre et al 2020).…”