Understanding PCR Processes to Draw Meaningful Conclusions from Environmental DNA Studies

Abstract

As environmental DNA (eDNA) studies have grown in popularity for use in ecological applications, it has become clear that their results differ in significant ways from those of traditional, non-PCR-based surveys. In general, eDNA studies that rely on amplicon sequencing may detect hundreds of species present in a sampled environment, but the resulting species composition can be idiosyncratic, reflecting species' true biomass abundances poorly or not at all. Here, we use a set of simulations to develop a mechanistic understanding of the processes leading to eDNA results. In particular, we focus on the effects of PCR cycle number and primer amplification efficiency on the results of diversity metrics in sequencing studies. We then show that proportional indices of eDNA reads capture trends in taxon biomass with high accuracy, particularly where amplification efficiency is high (median correlation up to 0.97). Our results explain much of the observed behavior of eukaryotic eDNA studies, and lead to recommendations for best practices in the field.

Integrating eDNA metabarcoding and simultaneous underwater visual surveys to describe complex fish communities in a marine biodiversity hotspot

“…was not detected (Hoplopagrus guenterii). To avoid this issue, a prospective evaluation of the taxonomic biases of the primers used in the context of the local fauna of interest, both in silico and in vitro, can help to experimentally assess possible primer mismatches (Kelly et al, 2019;Lecaudey et al, 2019). Additionally, a second barcode (e.g., 16S) analysed in parallel can support species identification and therefore, detection (Alberdi et al, 2017;Miya et al, 2015).…”

“…However, increasing the number of PCR cycles can also introduce PCR artefacts, and limiting cycles (particularly during PCR2) can improve the quality of the data (Kelly et al, 2019). Therefore, a balance in these parameters, as to not substantially increase PCR amplification errors during this step, is recommended (Ficetola et al, 2014).…”

Environmental DNA provides quantitative estimates of Pacific hake abundance and distribution in the open ocean

“…visual [13,14], capture [15,16] or acoustic [12] surveys) and the degree of agreement between individual samples of eDNA and traditional methods collected simultaneously often determines whether eDNA-based methods are viewed as successful or not [12,16]. However, eDNA observations arise from fundamentally different processes than observations from these traditional survey methods-most dramatically, by exponential amplification of DNA molecules in an environmental sample [17,18], but also because the distribution of eDNA itself in the environment is not identical to the distribution of its source organisms [5,7,8]. In the case of microbial eDNA, this distributional distinction is negligible, but for larger animals-such as fishes or marine mammals-it is not.…”

Environmental DNA metabarcoding reveals winners and losers of global change in coastal waters

“…By treating amplification efficiency as consistent within a given taxon, we created an index of abundance for each taxon across space and time ('eDNA index' [37]), using pooled data from technical replicates and mean proportions across biological replicates. We used this index of abundance in the multivariate community analysis, and used binary ( presence/absence) data to capture species-level responses to environmental conditions.…”

“…(b) Most-likely cluster as a function of temperature and pH given a multinomial logistic model (salinity was uninformative, being tightly correlated with temperature). (c) Relative abundance (eDNA index; see[37]) of the taxa best distinguishing the three communities illustrated (SIMPER analysis). See electronic supplementary material for full analysis.…”