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Understanding PCR Processes to Draw Meaningful Conclusions from Environmental DNA Studies.

ABSTRACT: As environmental DNA (eDNA) studies have grown in popularity for use in ecological applications, it has become clear that their results differ in significant ways from those of traditional, non-PCR-based surveys. In general, eDNA studies that rely on amplicon sequencing may detect hundreds of species present in a sampled environment, but the resulting species composition can be idiosyncratic, reflecting species' true biomass abundances poorly or not at all. Here, we use a set of simulations to develop a mechanistic understanding of the processes leading to the kinds of results common in mixed-template PCR-based (metabarcoding) studies. In particular, we focus on the effects of PCR cycle number and primer amplification efficiency on the results of diversity metrics in sequencing studies. We then show that proportional indices of amplicon reads capture trends in taxon biomass with high accuracy, particularly where amplification efficiency is high (median correlation up to 0.97). Our results explain much of the observed behavior of PCR-based studies, and lead to recommendations for best practices in the field.

SUBMITTER: Kelly RP

PROVIDER: S-EPMC6702206 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Understanding PCR Processes to Draw Meaningful Conclusions from Environmental DNA Studies.

Kelly Ryan P RP Shelton Andrew Olaf AO Gallego Ramón R

Scientific reports 20190820 1

As environmental DNA (eDNA) studies have grown in popularity for use in ecological applications, it has become clear that their results differ in significant ways from those of traditional, non-PCR-based surveys. In general, eDNA studies that rely on amplicon sequencing may detect hundreds of species present in a sampled environment, but the resulting species composition can be idiosyncratic, reflecting species' true biomass abundances poorly or not at all. Here, we use a set of simulations to d ...[more]

PMID: 31431641

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Project description:Aims:It is generally accepted that post-MI heart failure (HF) changes a variety of aspects of sarcoplasmic reticular Ca2+ fluxes but for some aspects there is disagreement over whether there is an increase or decrease. The commonest statistical approach is to treat data collected from each cell as independent, even though they are really clustered with multiple likely similar cells from each heart. In this study, we test whether this statistical assumption of independence can lead the investigator to draw conclusions that would be considered erroneous if the analysis handled clustering with specific statistical techniques (hierarchical tests). Methods and results:Ca2+ transients were recorded in cells loaded with Fura-2AM and sparks were recorded in cells loaded with Fluo-4AM. Data were analysed twice, once with the common statistical approach (assumption of independence) and once with hierarchical statistical methodologies designed to allow for any clustering. The statistical tests found that there was significant hierarchical clustering. This caused the common statistical approach to underestimate the standard error and report artificially small P values. For example, this would have led to the erroneous conclusion that time to 50% peak transient amplitude was significantly prolonged in HF. Spark analysis showed clustering, both within each cell and also within each rat, for morphological variables. This means that a three-level hierarchical model is sometimes required for such measures. Standard statistical methodologies, if used instead, erroneously suggest that spark amplitude is significantly greater in HF and spark duration is reduced in HF. Conclusion:Ca2+ fluxes in isolated cardiomyocytes show so much clustering that the common statistical approach that assumes independence of each data point will frequently give the false appearance of statistically significant changes. Hierarchical statistical methodologies need a little more effort, but are necessary for reliable conclusions. We present cost-free simple tools for performing these analyses.

Project description:BackgroundWe aimed to compare the efficacy and safety of bivalirudin versus heparin as the anticoagulant in patients undergoing extracorporeal membrane oxygenation (ECMO).MethodsWe conducted a search in PubMed, Embase and the Cochrane Library for all the studies in which bivalirudin was compared to heparin as the anticoagulant for ECMO. Efficacy outcomes were defined as the time to reach therapeutic levels, time within therapeutic range (TTR), thrombotic events, circuit thrombosis, circuit exchanges. Safety outcomes were reported as heparin-induced thrombocytopenia (HIT), major bleeding events, minor bleeding events. Other outcomes included hospital length of stay (LOS), ICU LOS, mortality, 30-day mortality and in-hospital mortality.ResultsTen studies with 1091 patients were included for meta-analysis. A significant reduction in thrombotic events [OR 0.51, 95%CI 0.36,0.73, p = 0.0002, I2 = 0%], major bleeding events [OR 0.31, 95%CI 0.10,0.92, p = 0.04, I2 = 75%] and in-hospital mortality [OR 0.63, 95%CI 0.44,0.89, p = 0.009, I2 = 0%] treated with bivalirudin were found compared with heparin. There were no significant differences between groups regarding the time to reach therapeutic levels [MD 3.53, 95%CI -4.02,11.09, p = 0.36, I2 = 49%], TTR [MD 8.64, 95%CI -1.72,18.65, p = 0.10, I2 = 77%], circuit exchanges [OR 0.92, 95%CI 0.27,3.12, p = 0.90, I2 = 38%], HIT [OR 0.25, 95%CI 0.02,2.52, p = 0.24, I2 = 0%], minor bleeding events [OR 0.93, 95%CI 0.38,2.29, p = 0.87, I2 = 0%], hospital LOS [MD -2.93, 95%CI -9.01,3.15, p = 0.34, I2 = 45%], ICU LOS [MD -4.22, 95%CI -10.07,1.62, p = 0.16, I2 = 0%], mortality [OR 1.84, 95%CI 0.58,5.85, p = 0.30, I2 = 60%] and 30-day mortality [OR 0.75, 95%CI 0.38,1.48, p = 0.41, I2 = 0%].ConclusionBivalirudin probably be a potential choice for ECMO anticoagulation. However, based on the included studies' limitation, the superiority of bivalirudin over heparin for anticoagulation in the ECMO population still require further prospective randomized controlled studies before a definite conclusion.

Project description:BACKGROUND:Systematic reviews are increasingly prevalent in environmental health due to their ability to synthesize evidence while reducing bias. Different systematic review methods have been developed by the US National Toxicology Program's Office of Health Assessment and Translation (OHAT), the US Environmental Protection Agency's (EPA) Integrated Risk Information System (IRIS), and by the US EPA under the Toxic Substances Control Act (TSCA), including the approach to assess risk of bias (ROB), one of the most vital steps which is used to evaluate internal validity of the studies. Our objective was to compare the performance of three tools (OHAT, IRIS, TSCA) in assessing ROB. METHODS:We selected a systematic review on polybrominated diphenyl ethers and intelligence quotient and/or attention deficit hyperactivity disorder because it had been endorsed by the National Academy of Sciences. Two reviewers followed verbatim instructions from the tools and independently applied each tool to assess ROB in 15 studies previously identified. We documented the time to apply each tool and the impact the ROB ratings for each tool had on the final rating of the quality of the overall body of evidence. RESULTS:The time to complete the ROB assessments varied widely (mean = 20, 32, and 40?min per study for the OHAT, IRIS, and TSCA tools, respectively). All studies were rated overall "low" or "uninformative" using IRIS, due to "deficient" or "critically deficient" ratings in one or two domains. Similarly, all studies were rated "unacceptable" using the TSCA tool because of one "unacceptable" rating in a metric related to statistical power. Approximately half of the studies had "low" or "probably low ROB" ratings across all domains with the OHAT and Navigation Guide tools. CONCLUSIONS:Tools that use overall ROB or study quality ratings, such as IRIS and TSCA, may reduce the available evidence to assess the harms of environmental exposures by erroneously excluding studies, which leads to inaccurate conclusions about the quality of the body of evidence. We recommend using ROB tools that circumvents these issues, such as OHAT and Navigation Guide. SYSTEMATIC REVIEW REGISTRATION:This review has not been registered as it is not a systematic review.

Project description:Environmental enteric dysfunction (EED), a chronic diffuse inflammation of the small intestine, is associated with stunting in children in the developing world. The pathobiology of EED is poorly understood because of the lack of a method to elucidate the host response. This study utilized a novel microarray method to interrogate the host transcriptome in feces in Malawian children with EED. Our data showed that the children studied had a range of %L values, consistent a spectrum of EED from normal to severe. We identified 12 transcripts associated with the severity of EED, including chemokines that stimulate T-cell proliferation, Fc fragments of multiple immunoglobulin families, interferon-induced proteins, activators of neutrophils and B-cells, and mediators that dampen cellular responses to hormones. EED associated transcripts mapped to pathways related to cell adhesion, and responses to a broad spectrum of viral, bacterial and parasitic microbes and enhanced phagocytosis. Several mucins, regulatory factors and protein kinases associated with the maintenance of the mucous layer were expressed less in children with EED than normal children. In conclusion, EED represents the focused activation of elements of the immune system and is associated with widespread intestinal barrier disruption. The differentially expressed transcripts may be explored as potential biomarkers. This study was carried out, firstly using Affymetrix Human Transcriptome Array 2.0 (HTA2.0, data have already been deposited in the GEO database: GSE74681) to profile the transcriptome of the host stool RNAs in 259 subjects, and then using droplet digital PCR (ddPCR) assays to validate the microarray data in terms of signal correlation. The ddPCR assay was done for a total of 60 genes in 25 - 236 (median 67) of the 258 RNA samples per RNA availability (1 of 259 samples [stool_080B] was not used for any of the 60 assays due to lack of availability). The data of the first 42 assays (genes) in the matrix sheets were used in Figure 4 for signal correlation analysis, and the data of the rest 18 assays (genes) were used in Table 4 for validation of 18 significant genes identified in biological pathway analysis.

Project description:BackgroundWhen healthcare budgets are exogenous, cost-effectiveness thresholds (CETs) used to inform funding decisions should represent the health opportunity cost (HOC) of such funding decisions, but HOC-based CET estimates have not been available until recently. In recent years, empirical HOC-based CETs for multiple countries have been published, but the use of these CETs in the cost-effectiveness analysis (CEA) literature has not been investigated. Analysis of the use of HOC-based CETs by researchers undertaking CEAs in countries with different decision-making contexts will provide valuable insights to further understand barriers and facilitators to the acceptance and use of HOC-based CETs.ObjectivesWe aimed to identify the CET values used to interpret the results of CEAs published in the scientific literature before and after the publication of jurisdiction-specific empirical HOC-based CETs in four countries.MethodsWe undertook a scoping review of CEAs published in Spain, Australia, the Netherlands and South Africa between 2016 (2014 in Spain) and 2020. CETs used before and after publication of HOC estimates were recorded. We conducted logit regressions exploring factors explaining the use of HOC values in identified studies and linear models exploring the association of the reported CET value with study characteristics and results.Results1171 studies were included in this review (870 CEAs and 301 study protocols). HOC values were cited in 28% of CEAs in Spain and in 11% of studies conducted in Australia, but they were not referred to in CEAs undertaken in the Netherlands and South Africa. Regression analyses on Spanish and Australian studies indicate that more recent studies, studies without a conflict of interest and studies estimating an incremental cost-effectiveness ratio (ICER) below the HOC value were more likely to use the HOC as a threshold reference. In addition, we found a small but significant impact indicating that for every dollar increase in the estimated ICER, the reported CET increased by US$0.015. Based on the findings of our review, we discuss the potential factors that might explain the lack of adoption of HOC-based CETs in the empirical CEA literature.ConclusionsThe adoption of HOC-based CETs by identified published CEAs has been uneven across the four analysed countries, most likely due to underlying differences in their decision-making processes. Our results also reinforce a previous finding indicating that CETs might be endogenously selected to fit authors' conclusions.

Dataset Information

Understanding PCR Processes to Draw Meaningful Conclusions from Environmental DNA Studies.

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Understanding PCR Processes to Draw Meaningful Conclusions from Environmental DNA Studies.

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