Parallel, targeted analysis of environmental samples via high‐throughput quantitative PCR

Abstract

Environmental DNA (eDNA) sampling in aquatic environments is a unique tool in that the taxonomic coverage represented by a single sample is large, with virtually all members of the local biota potentially represented (Hauck, Weitemeir, Penaluna, Garcia, & Cronn, 2019). The obvious potential of eDNA sampling to revolutionize monitoring, bioassessment, and ecological research has been pointed out repeatedly in recent years (Cristescu & Hebert, 2018; Deiner et al., 2017). However, accessing taxonomically broad biodiversity information from environmental samples is nontrivial. The available eDNA analysis methods present an apparent tradeoff between taxonomic scope and reliability of detection. One end of the spectrum is represented by single-taxon methods (e.g., taxon-specific quantitative or digital PCR). Properly designed taxon-specific assays are free from cross-amplification with other taxa and can reliably detect target DNA at low concentrations (e.g., <10 copies per reaction; Klymus et al., 2019). These approaches necessarily have a narrow taxonomic scope, but also tend to be more sensitive for rare species detection than multi-taxa approaches (Harper et al., 2018;

Better boundaries: identifying the upper extent of fish distributions in forested streams using eDNA and electrofishing

“…In particular, eDNA may be useful in streams where habitat is difficult to effectively sample using electrofishing because of either complex structures or upstream wetlands or when eggs are in the ground. Also, eDNA techniques will need to permit that managers target all possible fish species in a stream (e.g., parallel analyses of eDNA samples through high‐throughput PCR [Wilcox et al 2020] or use next‐generation sequencing) so that streams are not mistakenly classified as fishless. Future studies can focus on understanding fish movement at their upper extent across seasons, redefine barriers to fish movement, and determine the contribution of these headwater fishes to the overall fish population to further elucidate their importance to overall population dynamics.…”

Simultaneous absolute quantification and sequencing of fish environmental DNA in a mesocosm by quantitative sequencing technique

“…Quantification by qPCR or dPCR requires an assay to be established for each target, including the design of PCR primers, preparation of standards (if qPCR is used), and optimisation of PCR conditions. Therefore, parallel quantitative analysis of numerous species is not straightforward, although it has been recently attempted 14 .…”

Endpoint PCR coupled with capillary electrophoresis (celPCR) provides sensitive and quantitative measures of environmental DNA in singleplex and multiplex reactions

“…If target DNA is expected to be present mostly at concentrations below 10 copies per µl extract (i.e. 10 copies per 10 µl PCR reaction volume), it is, however, possible to pre-amplify target DNA with a preceding singleplex PCR targeting for example all fish DNA contained in a sample [39]. Our results were consistent between dPCR, and multiplex celPCR, except for two field-collected samples, which tested negative in multiplex celPCR, but contained < 0.25 copies per µl extract in dPCR.…”

“…Albeit distinction via fragment length differences is also possible for qPCR and dPCR [36,37], multiplexes on these instruments frequently employ specific dyes (attached to the respective probes) for each target [34,37]. The limited number of available dyes and their potential influence on primer/probe properties in addition to of all the above mentioned factors [17,38], make the development of endpoint PCR / celPCR multiplexes more feasible in comparison to qPCR and dPCR (but see [39] for a high-throughput qPCR approach). Generally, the use of multiplex PCRs enhances the cost- and time-effectiveness of any screening for specific target taxa [17,27,34], but there has been no in-depth assessment whether this is possible without forfeiting sensitivity and whether it is truly beneficial compared to singleplex endpoint PCRs, qPCRs, and dPCRs, which are most commonly applied for the detection of individual taxa from environmental samples.…”