Targeted next‐generation sequencing of environmental DNA improves detection of invasive European green crab (Carcinus maenas)

Abstract

In the northeast Pacific Ocean, there is high interest in developing eDNA‐based survey methods to aid management of invasive populations of European green crab (Carcinus maenas). Expected benefits are improved sensitivity for early detection of secondary spread and to assess the outcome of eradication efforts. A new eDNA‐based approach we term “targeted next‐generation sequencing (tNGS)” is introduced here and shown to improve detection relative to qPCR at sites with lower green crab CPUE values measured by trapping. DNA standards (gBlock) with starting molecule copies that were 10 to 100 times lower than the qPCR limit of detection returned significant numbers of sequencing reads, which in our field assessments translated to a 7%–10% increase in detection probability from tNGS relative to qPCR at sites with lower CPUE. We also found the number of sequencing reads from tNGS was significantly correlated with green crab CPUE whereas Ct values from qPCR were not. When sources of variation were partitioned for each assay, we found the difference between mean within‐site and mean between‐site variation was much larger and had non‐overlapping confidence intervals for tNGS relative to qPCR, suggesting the former may offer more power for detecting spatial variation in eDNA availability. Results presented here indicate this approach is suitable for species of known low abundances where a positive detection has high economic or environmental consequences, or for labs doing eDNA surveys for whom NGS‐based workflows are operationally more efficient than qPCR given its upward scalability. Any species with an existing qPCR assay can be easily tested with a tNGS assay using the approach presented here. We conclude with a discussion on the fitness for purpose of tNGS vs. qPCR for various applications and on how to best apply molecular surveys in management programs.

The Application of eDNA for Monitoring Aquatic Non-Indigenous Species: Practical and Policy Considerations

“…Although it is less common and opposes the term 'passive surveillance', a metabarcoding approach using primers for predetermined taxa groups (order or family level) can also be used to reduce the number of non-NIS present in the output datasets. In fact, Westfall, et al (2022) [62] used targeted NGS (tNGS) to improve early detection of invasive populations of the European green crab. By selectively targeting gene regions for a specific group of taxa, high sample volumes can still be processed while potentially increasing taxonomic resolution compared to whole community-based approaches.…”

“…The number of DNA copies can then be used as a proxy for the number of individuals, but this relationship can vary depending on factors such as the number of gene copies per individual and the eDNA shedding rate in the environment [72]. For the 'passive approach', several studies have also found a correlation between biomass and read counts derived from HTS of eDNA samples [62,73]. Interestingly, Westfall, et al (2022) [62] found that the number of sequencing reads from targeted NGS (tNGS) was significantly correlated with the invasive green crab catch per unit effort (CPUE), whereas Ct values from qPCR were not.…”

“…For the 'passive approach', several studies have also found a correlation between biomass and read counts derived from HTS of eDNA samples [62,73]. Interestingly, Westfall, et al (2022) [62] found that the number of sequencing reads from targeted NGS (tNGS) was significantly correlated with the invasive green crab catch per unit effort (CPUE), whereas Ct values from qPCR were not. The authors suggested that tNGS may offer more power for detecting spatial variation in eDNA availability particularly for early detection, making it suitable for species of known low abundances where a positive detection could have high economic or environmental consequences.…”

Comparative analysis of fish environmental DNA reveals higher sensitivity achieved through targeted sequence‐based metabarcoding

“…However, most of these comparative analyses had two to four times more technical replicates for qPCR than for metabarcoding, and thus analysed more total input DNA for qPCR (Bylemans et al, 2019; Harper et al, 2018; Lacoursière‐Roussel, Dubois, et al, 2016; Schenekar et al, 2020). A recent study comparing two targeted approaches found that NGS had a higher sensitivity than qPCR for the detection of invasive green crab (Westfall et al, 2021). Rare species detection from eDNA using NGS can also be improved by using higher sequencing depths, which have been facilitated by recent technical advances in sequencing technologies (Reinholdt Jensen et al, 2021; Singer et al, 2019).…”

“…A recent study comparing two targeted approaches found that NGS had a higher sensitivity than qPCR for the detection of invasive green crab (Westfall et al, 2021). Rare species detection from eDNA using NGS can also be improved by using higher sequencing depths, which have been facilitated by recent technical advances in sequencing technologies (Reinholdt Jensen et al, 2021;Singer et al, 2019).…”