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Implementation of Novel Design Features for qPCR-Based eDNA Assessment.

ABSTRACT: Environmental stewardship requires timely, accurate information related to the status of a given ecosystem and the species that occupy it. Recent advances in the application of the highly sensitive real-time quantitative polymerase chain reaction (qPCR) towards identification of constituents within environmental DNA (eDNA) now allow targeted detection of the presence of species-specific biological material within a localized geographic region. However, as with all molecular techniques predicated on the specificity and sensitivity of the PCR assay, careful validation of each eDNA qPCR assay in development must be performed both under controlled laboratory conditions and when challenged with field-derived eDNA samples. Such a step-wise approach forms the basis for incorporation of innovative qPCR design features that strengthen the implementation and interpretation of the eDNA assay. This includes empirical determination that the qPCR assay is refractory to the presence of human DNA and the use of a tripartite assay approach comprised of 1) a primer set targeting plant chloroplast that evaluates the presence of amplifiable DNA from field samples to increase confidence in a negative result, 2) an animal group primer set to increase confidence in the assay result, and 3) a species-specific primer set to assess presence of DNA from the target species. To demonstrate this methodology, we generated eDNA assays specific for the North American bullfrog (Lithobates (Rana) catesbeiana) and the Rocky Mountain tailed frog (Ascaphus montanus) and characterized each with respect to detection sensitivity and specificity with demonstrated performance in a field survey scenario. The qPCR design features presented herein address specific challenges of eDNA assays thereby increasing their interpretative power.

SUBMITTER: Veldhoen N

PROVIDER: S-EPMC5089736 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Implementation of Novel Design Features for qPCR-Based eDNA Assessment.

Veldhoen Nik N Hobbs Jared J Ikonomou Georgios G Hii Michael M Lesperance Mary M Helbing Caren C CC

PloS one 20161101 11

Environmental stewardship requires timely, accurate information related to the status of a given ecosystem and the species that occupy it. Recent advances in the application of the highly sensitive real-time quantitative polymerase chain reaction (qPCR) towards identification of constituents within environmental DNA (eDNA) now allow targeted detection of the presence of species-specific biological material within a localized geographic region. However, as with all molecular techniques predicated ...[more]

PMID: 27802293

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Project description:Global biodiversity is declining at an ever-increasing rate. Yet effective policies to mitigate or reverse these declines require ecosystem condition data that are rarely available. Morphology-based bioassessment methods are difficult to scale, limited in scope, suffer prohibitive costs, require skilled taxonomists, and can be applied inconsistently between practitioners. Environmental DNA (eDNA) metabarcoding offers a powerful, reproducible and scalable solution that can survey across the tree-of-life with relatively low cost and minimal expertise for sample collection. However, there remains a need to condense the complex, multidimensional community information into simple, interpretable metrics of ecological health for environmental management purposes. We developed a riverine taxon-independent community index (TICI) that objectively assigns indicator values to amplicon sequence variants (ASVs), and significantly improves the statistical power and utility of eDNA-based bioassessments. The TICI model training step uses the Chessman iterative learning algorithm to assign health indicator scores to a large number of ASVs that are commonly encountered across a wide geographic range. New sites can then be evaluated for ecological health by averaging the indicator value of the ASVs present at the site. We trained a TICI model on an eDNA dataset from 53 well-studied riverine monitoring sites across New Zealand, each sampled with a high level of biological replication (n = 16). Eight short-amplicon metabarcoding assays were used to generate data from a broad taxonomic range, including bacteria, microeukaryotes, fungi, plants, and animals. Site-specific TICI scores were strongly correlated with historical stream condition scores from macroinvertebrate assessments (macroinvertebrate community index or MCI; R2 = 0.82), and TICI variation between sample replicates was minimal (CV = 0.013). Taken together, this demonstrates the potential for taxon-independent eDNA analysis to provide a reliable, robust and low-cost assessment of ecological health that is accessible to environmental managers, decision makers, and the wider community.

Project description:BackgroundImplementation research is increasingly being recognised for optimising the outcomes of clinical practice. Frequently, the benefits of new evidence are not implemented due to the difficulties applying traditional research methodologies to implementation settings. Randomised controlled trials are not always practical for the implementation phase of knowledge transfer, as differences between individual and organisational readiness for change combined with small sample sizes can lead to imbalances in factors that impede or facilitate change between intervention and control groups. Within-cluster repeated measure designs could control for variance between intervention and control groups by allowing the same clusters to receive a sequence of conditions. Although in implementation settings, they can contaminate the intervention and control groups after the initial exposure to interventions. We propose the novel application of counterbalanced design to implementation research where repeated measures are employed through crossover, but contamination is averted by counterbalancing different health contexts in which to test the implementation strategy.MethodsIn a counterbalanced implementation study, the implementation strategy (independent variable) has two or more levels evaluated across an equivalent number of health contexts (e.g. community-acquired pneumonia and nutrition for critically ill patients) using the same outcome (dependent variable). This design limits each cluster to one distinct strategy related to one specific context, and therefore does not overburden any cluster to more than one focussed implementation strategy for a particular outcome, and provides a ready-made control comparison, holding fixed. The different levels of the independent variable can be delivered concurrently because each level uses a different health context within each cluster to avoid the effect of treatment contamination from exposure to the intervention or control condition.ResultsAn example application of the counterbalanced implementation design is presented in a hypothetical study to demonstrate the comparison of 'video-based' and 'written-based' evidence summary research implementation strategies for changing clinical practice in community-acquired pneumonia and nutrition in critically ill patient health contexts.ConclusionA counterbalanced implementation study design provides a promising model for concurrently investigating the success of research implementation strategies across multiple health context areas such as community-acquired pneumonia and nutrition for critically ill patients.

Dataset Information

Implementation of Novel Design Features for qPCR-Based eDNA Assessment.

Publications

Implementation of Novel Design Features for qPCR-Based eDNA Assessment.

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