The need for robust qPCR‐based eDNA detection assays in environmental monitoring and species inventories

Langlois, Valérie S.; Allison, M. J.; Bergman, Lauren C.; To, Tuan Anh; Helbing, Caren C.

doi:10.1002/edn3.164

Cited by 66 publications

(71 citation statements)

References 42 publications

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“…laboratory tests with reference tissue samples) and in situ validation (i.e. field tests with eDNA samples) (see Goldberg et al, 2016; MacDonald & Sarre, 2017; Langlois et al, 2020; So, Fong, Lam, & Dudgeon, 2020). Understanding the utility of an assay requires both knowledge of the context in which it has been designed, and a broader understanding of how it was developed.…”

Section: Criteria and Principles Of Validationmentioning

confidence: 99%

“…Generally, these values are obtained using a dilution series of quantified DNA amplicons or synthesized gene fragments (e.g. IDT gBlocks™ Gene Fragment) based on public or de novo reference sequences (Langlois et al, 2020). The LOD and LOQ have various definitions in the eDNA literature, but were recently standardised by Klymus et al (2019), where LOD is the lowest standard concentration at which 95% of technical replicates amplify and LOQ is the lowest standard concentration for which the coefficient of variation (CV) value is <35%.…”

Section: Criteria and Principles Of Validationmentioning

confidence: 99%

“…Within the last decade, species detection from environmental DNA (eDNA; i.e. detection of extra-organismal DNA released by organisms into their environment) has shown great potential for routine species surveys (Rees, Maddison, Middleditch, Patmore, & Gough, 2014; Goldberg et al, 2016; Deiner et al, 2017; Langlois, Allison, Bergman, To, & Helbing, 2020; Sepulveda, Nelson, Jerde, & Luikart, 2020). The interest in molecular species detection has fuelled the development of over 500 assays, reviewed herein, that utilise PCR to amplify DNA or RNA extracted from environmental samples.…”

Section: Introductionmentioning

confidence: 99%

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A validation scale to determine the readiness of environmental DNA assays for routine species monitoring

Thalinger

Deiner

Harper

et al. 2020

Preprint

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32 33 1. Environmental DNA (eDNA) analysis utilises trace DNA released by organisms into their 34 environment for species detection and is revolutionising non-invasive species monitoring. The 35 use of this technology requires rigorous validation -from field sampling to interpretation of PCR-36 based results -for meaningful application and interpretation. Assays targeting eDNA released by 37 individual species are typically validated with no predefined criteria to answer specific research 38 questions in one ecosystem. Their general applicability, uncertainties and limitations often 39 remain undetermined. The absence of clear guidelines prevents targeted eDNA assays from 40 being incorporated into species monitoring and policy, thus their establishment will be key for the 41 future implementation of eDNA-based surveys. 42 2. We describe the measures and tests necessary for successful validation of targeted eDNA 43 assays and the associated pitfalls to form the basis of guidelines. A list of 122 variables was 44 compiled and consolidated into a scale to assess the validation status of individual assays. 45These variables were evaluated for 546 published single-species assays. The resulting dataset 46 was used to provide an overview of current validation practices and test the applicability of the 47 (30%) of investigated assays were classified as Level 1 (incomplete), and 15% did not achieve 52 this first level. These assays were characterised by minimal in silico and in vitro testing, but their 53 share in annually published eDNA assays has declined since 2014. The total number of reported 54 variables ranged from 20% to 76% and deviated both between and within levels. 55 4. The meta-analysis demonstrates the suitability of the 5-level validation scale for assessing 56 targeted eDNA assays. It is a user-friendly tool to evaluate previously published assays for future 57 4 research and routine monitoring, while also enabling appropriate interpretation of results. Finally, 58 it provides guidance on validation and reporting standards for newly developed assays. 59 60

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Section: Criteria and Principles Of Validationmentioning

confidence: 99%

Section: Criteria and Principles Of Validationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A validation scale to determine the readiness of environmental DNA assays for routine species monitoring

Thalinger

Deiner

Harper

et al. 2020

Preprint

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“…faeces, excretion, shedding cells, gametes) and this eDNA can take different forms (tissues, cells, organites, nucleo-proteic complexes, …). The direct sampling of eDNA coupled with molecular analysis methods such as NGS (Shokralla et al, 2012) or quantitative polymerase chain reaction (qPCR) (Langlois et al, 2020) allow the detection and identification of aquatic species while overcoming organism capture. Although eDNA offers many promising applications, several methodological challenges remain.…”

Section: Introductionmentioning

confidence: 99%

Passive sampling of environmental DNA in aquatic environments using 3D-printed hydroxyapatite samplers

Verdier

Konecny‐Dupré

Marquette

et al. 2021

Preprint

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1. The study of environmental DNA released by aquatic organisms in their habitat offers a fast, non-invasive and sensitive approach to monitor their presence. Common eDNA sampling methods such as filtration and precipitation are time consuming, require human intervention and are not applicable to a wide range of habitats such as turbid waters and poorly-accessible environments. To circumvent these limitations, we propose to use the binding properties of minerals to create a passive eDNA sampler. 2. We have designed 3D-printed samplers made of hydroxyapatite (HAp samplers), a mineral known for its high binding affinity with DNA. The shape and the geometry of the samplers have been designed to facilitate their handling in laboratory and field. Here we describe and test the ability of HAp samplers to recover artificial DNA and eDNA. 3. We show that HAp samplers efficiently recover DNA and are effective even on small amounts of eDNA (<1 ng). However, we also observed large variations in the amount of DNA recovered even under controlled conditions. 4. By better understanding the physico-chemical interactions between DNA and the HAp sampler surface, one could improve the repeatability of the sampling process and provide an easy-to-use eDNA sampling tool for aquatic environments.

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“…Decreased precision at low eDNA concentrations is also linked with intrinsic technical limitations of quantitative PCR methods such as quantitative (q)PCR or digital droplet (d)PCR, the former being the most common method for targeted species detection and quantification. False positive fluorescent background signals (Strain et al, 2013) and contamination from non-specific amplification can lead to over-estimation of eDNA at low concentrations (Hunter et al, 2018; Langlois et al, 2020), such as would be expected from rare species eDNA. Stochastic variation of molecule distribution in an aqueous sample and the propensity of DNA to adsorb onto plastic tube walls and pipette tips are known to affect the proportion of positive results from low template concentrations (Ellison et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Targeted Next Generation Sequencing of environmental DNA improves detection and quantification of invasive European green crab (Carcinus maenas)

Westfall

2021

Preprint

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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 quantification of abundances 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 low eDNA concentrations, as is characteristic of founding or spreading populations. tNGS is based on the premise that the number of NGS reads from non-normalized (i.e. equal volumes) targeted PCR amplicons will approximate the starting DNA amount. Standard DNA concentrations 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 crab detection probability from tNGS relative to qPCR at low abundances. We also found that eDNA concentration was highly correlated with crab abundance, as measured from traditional trapping methods, for both assays; however, tNGS data had greater precision and less error than qPCR. When partitioning the sources of variation in each assay we identified greater between-site variability for tNGS relative to qPCR, suggesting the former may offer more power for detecting spatial variation in eDNA concentration. When applying this assay in management programs, we suggest including a panel of eDNA samples from sites with trapping data as standards to estimate relative abundance at sites with no a priori information. Results presented here indicate the tNGS approach has great promise for surveillance of green crab and could easily be adopted for surveillance of any species of high interest to management, including endangered species, new incursions of invasive species, and species with low eDNA shedding rates. Pros and cons of this approach compared to qPCR are discussed.

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The need for robust qPCR‐based eDNA detection assays in environmental monitoring and species inventories

Cited by 66 publications

References 42 publications

A validation scale to determine the readiness of environmental DNA assays for routine species monitoring

A validation scale to determine the readiness of environmental DNA assays for routine species monitoring

Passive sampling of environmental DNA in aquatic environments using 3D-printed hydroxyapatite samplers

Targeted Next Generation Sequencing of environmental DNA improves detection and quantification of invasive European green crab (Carcinus maenas)

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