Needle in a haystack? A comparison of eDNA metabarcoding and targeted qPCR for detection of the great crested newt (Triturus cristatus)

Abstract

SummaryEnvironmental DNA (eDNA) analysis is a rapid, cost-effective, non-invasive biodiversity monitoring tool which utilises DNA left behind in the environment by organisms for species detection. The method is used as a species specific survey tool for rare or invasive species across a broad range of ecosystems. Recently, eDNA and ‘metabarcoding’ have been combined to describe whole communities rather than focusing on single target species. However, whether metabarcoding is as sensitive as targeted approaches for rare species detection remains to be evaluated. The great crested newt Triturus cristatus is a flagship pond species of international conservation concern and the first UK species to be routinely monitored using eDNA. We evaluate whether eDNA metabarcoding has comparable sensitivity to targeted real-time quantitative PCR (qPCR) for T. cristatus detection. Extracted eDNA samples (N = 532) were screened for T. cristatus by qPCR and analysed for all vertebrate species using High-Throughput Sequencing technology. With qPCR and a detection threshold of 1/12 positive qPCR replicates, newts were detected in 50% of ponds. Detection decreased to 32% when the threshold was increased to 4/12 positive qPCR replicates. With metabarcoding, newts were detected in 34% of ponds without a detection threshold, and in 28% of ponds when a threshold (0.028%) was applied. Therefore, qPCR provided greater detection than metabarcoding but metabarcoding detection with no threshold was equivalent to qPCR with a stringent detection threshold. The proportion of T. cristatus sequences in each sample was positively associated with the number of positive qPCR replicates (qPCR score) suggesting eDNA metabarcoding may be indicative of eDNA concentration. eDNA metabarcoding holds enormous potential for holistic biodiversity assessment and routine freshwater monitoring. We advocate this community approach to freshwater monitoring to guide management and conservation, whereby entire communities can be initially surveyed to best inform use of funding and time for species-specific surveys.

Rapid assessment of coral cover from environmental DNA in Hawai'i

“…Finally, as a holistic approach to surveying marine biodiversity, previous studies have established that metabarcoding of eDNA from filtered seawater remains unmatched in terms of its cost‐effectiveness and sensitivity (Bakker et al, ; Boussarie et al, ; Port et al, ; Stat et al, ; Valentini et al, ; Yamamoto et al, ). However, the results from this study indicate that metabarcoding can also be used under some circumstances as a rapid alternative to qPCR in determining abundance of multiple taxa (Hänfling et al, ; Harper et al, ; Piñol et al, ; Pont et al, ; Stoeckle et al, ; Thomsen et al, ; Ushio et al, ). Additionally, this technique may be applicable not only to tracking changes in coral cover, but also to track changes in community composition, as well as to detect cryptic taxa, or new recruits, on coral reefs that may otherwise be missed by traditional visual‐based survey methods.…”

“…Regardless of the reason why, our study suggests that target sequence length must be carefully considered when designing eDNA metabarcoding assays of community abundance, yet sequence length has received little attention in eDNA study design (Goldberg et al, ; Taberlet et al, ; Wilcox et al, ). eDNA metabarcoding approaches have primarily focused on amplifying short (~100 bp) sequences (Hänfling et al, ; Harper et al, ; Pont et al, ; Port et al, ; Stoeckle et al, ; Thomsen et al, ; Valentini et al, ) to increase detection rates in systems with (presumably) highly degraded eDNA (Taberlet et al, ). While estimates of coral cover are more precise with a particular pair of short sequence primers (16S in our case), assays designed to measure diversity may require different tools than those designed for abundance estimates.…”

“…qPCR has become the standard for detection of rare species (Lacoursière‐Roussel et al, ; Nevers et al, ; Wilcox et al, ), and can provide information about species abundances based on eDNA biomass (Tillotson et al, ; Yamamoto et al, ). However, qPCR is limited in that fewer species can be studied at a time with species‐specific primers, making community‐wide abundance data more technically challenging, costly, and time consuming to ascertain (Harper et al, ).…”

“…The disadvantage of metabarcoding with universal primers is that it typically creates uneven PCR amplification across taxa due to the varying quality of the match between primers and a diverse template DNA pool (Bista et al, ; Elbrecht & Leese, ; Murray et al, ; Piñol et al, ; Taberlet et al, ). In the middle of this spectrum are taxon‐specific primers, that, by targeting a narrow range of taxa sharing conserved PCR priming sites, have the potential to recover estimates of abundance (Hänfling et al, ; Harper et al, ; Pont et al, ; Stoeckle et al, ; Thomsen et al, ; Ushio et al, ) as well as to detect greater diversity (Bakker et al, ; Balasingham, Walter, Mandrak, & Heath, ; Boussarie et al, ; Port et al, ; Stat et al, ; Valentini et al, ; Yamamoto et al, ) by limiting amplification of unwanted groups and maximizing amplification of target taxa.…”

A performance evaluation of targeted eDNA and eDNA metabarcoding analyses for freshwater fishes

“…One well‐recognized bias in metabarcoding analyses is preferential amplification of a few target sequences, that is, those that are relatively abundant or have few primer‐template mismatches (Bylemans, Gleeson, Hardy, & Furlan, ; Elbrecht & Leese, ). Amplification biases can increase the chance of false‐negative detections (failure to detect a species even when it is present) compared to a targeted approach (Harper et al, ; Lacoursière‐Roussel, Dubois, Normandeau, Bernatchez, & Adamowicz, ; Schneider et al, ). Concerns also remain as to whether eDNA metabarcoding data can be interpreted in a quantitative manner.…”