scholarly journals The use of multiple markers and internal positive controls significantly improves species eDNA detection rates and data reliability

2021 ◽  
Vol 4 ◽  
Author(s):  
Rein Brys ◽  
Teun Everts ◽  
David Halfmaerten ◽  
Sabrina Neyrinck ◽  
Quentin Mauvisseau
Keyword(s):  
False Negative ◽  
Strong Support ◽  
Environmental Dna ◽  
Single Species ◽  
Detection Rates ◽  
Multiple Markers ◽  
Molecular Approaches ◽  
Detection Probabilities ◽  
Elusive Species ◽  
Positive Controls

In recent years, environmental DNA analyses became increasingly integrated to detect and monitor the presence and abundance of rare organisms, especially in inaccessible aquatic habitats. Although it is generally proven that detection probabilities of eDNA surveys exceed those obtained via conventional techniques, these molecular approaches are, however, also subjected to detection limitations and levels of uncertainty. Besides improvements that can be made in terms of sampling design, volumes of filtered water, and the effective quantity of DNA that is finally analysed, the sensitivity of eDNA surveys is inherently determined by the number of target eDNA copies suspended in the water column. Here we show that multiplexing different primer/probe assays for the same species, but targeting amplicons situated at different loci, is a surprisingly overlooked aspect that can substantially contribute to reduce these limitations and increase the sensitivity of single-species detections. By empirically testing a large number of natural eDNA samples via ddPCR, we reveal that the use of multiple markers can significantly lower the LOD and LOQ of rare and elusive species, such as the invasive American bullfrog and the endangered European weather loach in a variety of different water bodies, such as ponds, lakes, streams, canals, etc. Especially at very low eDNA concentrations of both target species, our results showed that analysing mulitple loci significantly increased detection probabilities and lowered stochasticity effects, and thus ultimately reduces PCR costs when analysed in multiplex. The validation and use of more than one assay taregtting a single species, may further increase the confidence of positive detections. Finally, we illustrate that the implementation of internal positive controls (IPC's), is an absolute must for accurate validation of eDNA workflows and reliable interpretation of the generated data. IPC’s not only help to track down degraded and inhibited samples, to avoid false-negative detections, it also offers insights into extraction efficiency, indispensable for accurate quantification of population densities. Overall, our findings provide strong support that the multiplexing of multiple markers on different loci in combination with the use of internal positive controls ensures increased detection rates at very low eDNA concentrations and generates more robust and reliable data.

scholarly journals An assay validation framework to compare and evaluate targeted environmental DNA assays for routine species monitoring

2021 ◽  
Vol 4 ◽  
Author(s):  
Bettina Thalinger ◽  
Kristy Deiner ◽  
Lynsey Harper ◽  
Helen Rees ◽  
Rosetta Blackman ◽  
...  
Keyword(s):  
Meta Analysis ◽  
Environmental Dna ◽  
Single Species ◽  
End Users ◽  
Specific System ◽  
Initial Development ◽  
Assay Validation ◽  
Non Invasive ◽  
Detection Probabilities ◽  
User Friendly

Environmental DNA (eDNA) analysis utilises trace DNA released by organisms into their environment for species detection and is revolutionising non ‐ invasive species and biodiversity monitoring. However, this technology requires rigorous validation along the whole workflow – from field sampling to statistical analysis – to ensure appropriate and meaningful interpretation of results. Targeted eDNA assays are often validated within a specific system and with particular aims, but without fulfilling predefined criteria. Consequently, their applicability beyond initial development often remains undetermined. Additionally, there tends to be poor understanding of the uncertainties and limitations associated with already published assays and thus potentially inappropriate interpretation of the results they produce. The lack of a “gold standard” limits the incorporation of targeted eDNA assays into species monitoring and policy making by end-users and is therefore key for the future implementation of eDNA-based surveys. Here, we present a framework (https://edna-validation.com/) and user-friendly criteria for the classification of assays, which is based on previous validation efforts. A 5 ‐ level assay validation scale (“incomplete” to “operational”) was defined by reviewing the current eDNA literature and conducting a meta-analysis on sampling, laboratory practices, detection limits, and detection probabilities. The so far published single species eDNA assays were reviewed for their performance in this new framework and we identified steps within the validation process that often remain untouched. Finally, we provide guidance for end ‐ users as to which criteria are most important for validation and suggest how results obtained from assays at different levels of the validation scale should be interpreted.

Effects of Previous Surgery on the Detection of Sentinel Nodes in Women With Vulvar Cancer

2011 ◽  
Vol 21 (9) ◽  
pp. 1679-1683 ◽  
Author(s):  
Tessa A. Ennik ◽  
David G. Allen ◽  
Ruud L.M. Bekkers ◽  
Simon E. Hyde ◽  
Peter T. Grant
Keyword(s):  
Vulvar Cancer ◽  
False Negative ◽  
False Negative Rate ◽  
Blue Dye ◽  
Previous Surgery ◽  
Detection Rates ◽  
Reliable Technique ◽  
Appearance Time ◽  
Negative Rate ◽  
Significant Difference

BackgroundThere is a growing interest to apply the sentinel node (SN) procedure in the treatment of vulvar cancer. Previous vulvar surgery might disrupt lymphatic patterns and thereby decrease SN detection rates, lengthen scintigraphic appearance time (SAT), and increase SN false-negative rate. The aims of this study were to evaluate the SN detection rates at the Mercy Hospital for Women in Melbourne and to investigate whether previous vulvar surgery affects SN detection rates, SAT, and SN false-negative rate.MethodsData on all patients with vulvar cancer who underwent an SN procedure (blue dye, technetium, or combined technique) from November 2000 to July 2010 were retrospectively collected.ResultsSixty-five SN procedures were performed. Overall detection rate was 94% per person and 80% per groin. Detection rates in the group of patients who underwent previous excision of the primary tumor were not lower compared with the group without previous surgery or with just an incisional biopsy. There was no statistical significant difference in SAT between the previous excision group and the other patients. None of the patients with a false-negative SN had undergone previous excision.ConclusionsResults indicate that previous excision of a primary vulvar malignancy does not decrease SN detection rates or increase SN false-negative rate. Therefore, the SN procedure appears to be a reliable technique in patients who have previously undergone vulvar surgery. Previous excision did not significantly lengthen SAT, but the sample size in this subgroup analysis was small.

scholarly journals Genotyping Error Detection Through Tightly Linked Markers

Genetics ◽  
2003 ◽  
Vol 164 (3) ◽  
pp. 1161-1173
Author(s):  
Guohua Zou ◽  
Deyun Pan ◽  
Hongyu Zhao
Keyword(s):  
Error Detection ◽  
Complex Disease ◽  
Disease Genes ◽  
Genotyping Error ◽  
Genotyping Errors ◽  
Detection Rates ◽  
Nuclear Families ◽  
Additional Information ◽  
Number Of Children ◽  
Multiple Markers

Abstract The identification of genotyping errors is an important issue in mapping complex disease genes. Although it is common practice to genotype multiple markers in a candidate region in genetic studies, the potential benefit of jointly analyzing multiple markers to detect genotyping errors has not been investigated. In this article, we discuss genotyping error detections for a set of tightly linked markers in nuclear families, and the objective is to identify families likely to have genotyping errors at one or more markers. We make use of the fact that recombination is a very unlikely event among these markers. We first show that, with family trios, no extra information can be gained by jointly analyzing markers if no phase information is available, and error detection rates are usually low if Mendelian consistency is used as the only standard for checking errors. However, for nuclear families with more than one child, error detection rates can be greatly increased with the consideration of more markers. Error detection rates also increase with the number of children in each family. Because families displaying Mendelian consistency may still have genotyping errors, we calculate the probability that a family displaying Mendelian consistency has correct genotypes. These probabilities can help identify families that, although showing Mendelian consistency, may have genotyping errors. In addition, we examine the benefit of available haplotype frequencies in the general population on genotyping error detections. We show that both error detection rates and the probability that an observed family displaying Mendelian consistency has correct genotypes can be greatly increased when such additional information is available.

scholarly journals Airborne environmental DNA metabarcoding detects more diversity, with less sampling effort, than a traditional plant community survey

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Mark D. Johnson ◽  
Mohamed Fokar ◽  
Robert D. Cox ◽  
Matthew A. Barnes
Keyword(s):  
Plant Community ◽  
Plant Communities ◽  
Traditional Method ◽  
Environmental Dna ◽  
Single Species ◽  
Community Survey ◽  
Sampling Effort ◽  
Sampling Campaign ◽  
Dna Metabarcoding ◽  
Human Health Impacts

Abstract Background Airborne environmental DNA (eDNA) research is an emerging field that focuses on the detection of species from their genetic remnants in the air. The majority of studies into airborne eDNA of plants has until now either focused on single species detection, specifically only pollen, or human health impacts, with no previous studies surveying an entire plant community through metabarcoding. We therefore conducted an airborne eDNA metabarcoding survey and compared the results to a traditional plant community survey. Results Over the course of a year, we conducted two traditional transect-based visual plant surveys alongside an airborne eDNA sampling campaign on a short-grass rangeland. We found that airborne eDNA detected more species than the traditional surveying method, although the types of species detected varied based on the method used. Airborne eDNA detected more grasses and forbs with less showy flowers, while the traditional method detected fewer grasses but also detected rarer forbs with large showy flowers. Additionally, we found the airborne eDNA metabarcoding survey required less sampling effort in terms of the time needed to conduct a survey and was able to detect more invasive species than the traditional method. Conclusions Overall, we have demonstrated that airborne eDNA can act as a sensitive and efficient plant community surveying method. Airborne eDNA surveillance has the potential to revolutionize the way plant communities are monitored in general, track changes in plant communities due to climate change and disturbances, and assist with the monitoring of invasive and endangered species.

scholarly journals Environmental DNA (eDNA) metabarcoding surveys extend the range of invasion for non-indigenous freshwater species in Eastern Europe.

2021 ◽  
Author(s):  
Gert-Jan Jeunen ◽  
Tatsiana Lipinskaya ◽  
Helen Gajduchenko ◽  
Viktoriya Golovenchik ◽  
Michail Moroz ◽  
...  
Keyword(s):  
Surface Water ◽  
Water Samples ◽  
False Negative ◽  
Simultaneous Detection ◽  
Environmental Dna ◽  
Reference Database ◽  
Indigenous Species ◽  
Freshwater Species ◽  
Traditional Approaches ◽  
Surface Water Samples

Active environmental DNA (eDNA) surveillance through species-specific amplification has shown increased sensitivity in the detection of non-indigenous species (NIS) compared to traditional approaches. When many NIS are of interest, however, active surveillance decreases in cost- and time-efficiency. Passive surveillance through eDNA metabarcoding takes advantage of the complex DNA signal in environmental samples and facilitates the simultaneous detection of multiple species. While passive eDNA surveillance has previously detected NIS, comparative studies are essential to determine the ability of eDNA metabarcoding to accurately describe the range of invasion for multiple NIS versus alternative approaches. Here, we surveyed twelve sites, covering nine rivers across Belarus for NIS with three different techniques, i.e., an ichthyological, hydrobiological, and eDNA survey, whereby DNA was extracted from 500 mL surface water samples and amplified with two 16S rRNA primer assays targeting the fish and macro-invertebrate biodiversity. Nine non-indigenous fish and ten non-indigenous sediment-living macro-invertebrates were detected by traditional surveys, while seven NIS eDNA signals were picked up, including four fish, one aquatic and two sediment-living macro-invertebrates. Passive eDNA surveillance extended the range of invasion further north for two invasive fish and identified a new NIS for Belarus, the freshwater jellyfish Craspedacusta sowerbii. False-negative detections for the eDNA survey could be attributed to (i) preferential amplification of aquatic over sediment-living macro-invertebrates from surface water samples and (ii) an incomplete reference database. The evidence provided in this study recommends the implementation of both molecular-based and traditional approaches to maximize the probability of early detection of non-native organisms.

scholarly journals Understanding environmental DNA detection probabilities: A case study using a stream-dwelling char Salvelinus fontinalis

2016 ◽  
Vol 194 ◽  
pp. 209-216 ◽  
Author(s):  
Taylor M. Wilcox ◽  
Kevin S. McKelvey ◽  
Michael K. Young ◽  
Adam J. Sepulveda ◽  
Bradley B. Shepard ◽  
...  
Keyword(s):  
Salvelinus Fontinalis ◽  
Dna Detection ◽  
Environmental Dna ◽  
Detection Probabilities

scholarly journals False‐negative detections from environmental DNA collected in the presence of large numbers of killer whales ( Orcinus orca )

2019 ◽  
Vol 1 (4) ◽  
pp. 316-328 ◽  
Author(s):  
Róisín Pinfield ◽  
Eileen Dillane ◽  
Anne Kathrine W. Runge ◽  
Alice Evans ◽  
Luca Mirimin ◽  
...  
Keyword(s):  
False Negative ◽  
Environmental Dna ◽  
Orcinus Orca ◽  
Killer Whales ◽  
Large Numbers

Comparison of Baseline, Bone-Subtracted, and Enhanced Chest Radiographs for Detection of Pneumothorax

2020 ◽  
pp. 084653712090885
Author(s):  
Fatemeh Homayounieh ◽  
Subba R. Digumarthy ◽  
Jennifer A. Febbo ◽  
Sherief Garrana ◽  
Chayanin Nitiwarangkul ◽  
...  
Keyword(s):  
False Negative ◽  
Tension Pneumothorax ◽  
Area Under The Curve ◽  
Ground Truth ◽  
Chest Radiographs ◽  
Detection Rates ◽  
X Ray ◽  
Institutional Review ◽  
Type Area ◽  
Chest X Ray

Purpose: To assess and compare detectability of pneumothorax on unprocessed baseline, single-energy, bone-subtracted, and enhanced frontal chest radiographs (chest X-ray, CXR). Method and Materials: Our retrospective institutional review board–approved study included 202 patients (mean age 53 ± 24 years; 132 men, 70 women) who underwent frontal CXR and had trace, moderate, large, or tension pneumothorax. All patients (except those with tension pneumothorax) had concurrent chest computed tomography (CT). Two radiologists reviewed the CXR and chest CT for pneumothorax on baseline CXR (ground truth). All baseline CXR were processed to generate bone-subtracted and enhanced images (ClearRead X-ray). Four radiologists (R1-R4) assessed the baseline, bone-subtracted, and enhanced images and recorded the presence of pneumothorax (side, size, and confidence for detection) for each image type. Area under the curve (AUC) was calculated with receiver operating characteristic analyses to determine the accuracy of pneumothorax detection. Results: Bone-subtracted images (AUC: 0.89-0.97) had the lowest accuracy for detection of pneumothorax compared to the baseline (AUC: 0.94-0.97) and enhanced (AUC: 0.96-0.99) radiographs ( P < .01). Most false-positive and false-negative pneumothoraces were detected on the bone-subtracted images and the least numbers on the enhanced radiographs. Highest detection rates and confidence were noted for the enhanced images (empiric AUC for R1-R4 0.96-0.99). Conclusion: Enhanced CXRs are superior to bone-subtracted and unprocessed radiographs for detection of pneumothorax. Clinical Relevance/Application: Enhanced CXRs improve detection of pneumothorax over unprocessed images; bone-subtracted images must be cautiously reviewed to avoid false negatives.

scholarly journals Three-dimensional camera anthropometry to assess risk of cephalopelvic disproportion-related obstructed labour in Ethiopia

2019 ◽  
Vol 9 (5) ◽  
pp. 20190036 ◽  
Author(s):  
Lorenzo Tolentino ◽  
Mahlet Yigeremu ◽  
Sisay Teklu ◽  
Shehab Attia ◽  
Michael Weiler ◽  
...  
Keyword(s):  
False Negative ◽  
Three Dimensional ◽  
False Negative Rate ◽  
Detection Rates ◽  
Cephalopelvic Disproportion ◽  
Obstructed Labour ◽  
Tape Measure ◽  
Negative Rate ◽  
Positive Rate ◽  
Tape Measurement

Cephalopelvic disproportion (CPD)-related obstructed labour requires delivery via Caesarean section (C/S); however, in low-resource settings around the world, facilities with C/S capabilities are often far away. This paper reports three low-cost tools to assess the risk of CPD, well before labour, to provide adequate time for referral and planning for delivery. We performed tape measurement- and three-dimensional (3D) camera-based anthropometry, using two 3D cameras (Kinect and Structure) on primigravida, gestational age ≥ 36 weeks, from Addis Ababa, Ethiopia. Novel risk scores were developed and tested to identify models with the highest predicted area under the receiver-operator characteristic curve (AUC), detection rate (true positive rate at a 5% false-positive rate, FPR) and triage rate (true negative rate at a 0% false-negative rate). For tape measure, Kinect and Structure, the detection rates were 53%, 61% and 64% (at 5% FPR), the triage rates were 30%, 56% and 63%, and the AUCs were 0.871, 0.908 and 0.918, respectively. Detection rates were 77%, 80% and 84% at the maximum J -statistic, which corresponded to FPRs of 10%, 15% and 11%, respectively, for tape measure, Kinect and Structure. Thus, tape measurement anthropometry was a very good predictor and Kinect and Structure anthropometry were excellent predictors of CPD risk.

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