Eco-HTTr
This space houses datasets associated with ecological high throughput transcriptomics research
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Comparison of whole transcriptome and targeted RNA sequencing for ecological high-throughput transcriptomics
In 2019, in anticipation of a new program of research in ecological high-throughput transcriptomics, US EPA organized a federal government challenge aimed at identifying and evaluating low cost, high-throughput, RNA sequencing technologies that could support the aims of the research. Through a competition open to individuals and non-governmental organizations worldwide, innovators were invited to demonstrate their solutions. Each responding solver was provided a set of nine pooled RNA samples from each of four species of aquatic organisms (n=36 samples total). Three independent Solver teams submitted data, and technology descriptions, for five different analysis methods. Each was evaluated according to a pre-defined scoring rubric that considered accuracy, precision, transcriptome coverage for each species, and commercial viability (cost per sample and throughput considerations). Among the five Solutions submitted, a targeted approach (TempO-Seq) that employed sentinel gene sets representing 5-11% of the whole transcriptome was ranked as the top solution. However, all submitted solutions were determined to be viable for the research and had specific strengths and weaknesses. In a follow up investigation, transcriptomic points of departure based on a sentinel gene set were generally found to fall within a factor of 10 or less of those based on whole transcriptome sequencing. Results support the conclusion that a wide range of sequencing technologies and approaches are suitable for the work. Detailed and transparent reporting of the approaches used will help support uptake in science-based decision-making.
The dataset contains the Solver-submitted count matrices; R code for analyses that supported judging; In silico subsets.
In vitro estrogenic activity of perfluorinated diols of different chain lengths
Per- and polyfluoroalkyl substances (PFAS) are a diverse class of synthetic fluorinated chemicals that are currently ubiquitous in the environment due to their diverse usage in both consumer and industrial products. To help prioritize data poor PFAS, previous work evaluated 142 PFAS using an in vitro screening platform. To further evaluate effectiveness of the assays in identifying PFAS capable of inducing estrogenic responses in fish our lab group conducted in vivo exposures using fathead minnows (Pimephales promelas). 1H,1H,8H,8H-perfluorooctane-1,8-diol [FC8-diol], 1H,1H,10H,10H-perfluorodecane-1,10-diol [FC10-diol], and 1H,1H,8H,8H-perfluoro-3,6-dioxaoctane-1,8-diol [FC8-DOD] were found to be estrogenic in fathead minnows. The data stored here builds off these studies and was aimed to examine the ability of similar PFAS, primarily PFAS with the same structure but different chain lengths, to induce estrogenic responses in vitro. One PFAS with a high similarity score to FC10-diol (the most potent of the tested PFAS) but different structure was also included. Presented here are the data from Indigo ER bioassays along with data from a rainbow trout liver slice assay which measured estrogen receptor-regulated gene expression. Data sheets include the raw data from the plate reader (presented in blue under luminescence) and resulting data (E2 equivalence) and figures from PRISM analysis. Data sheets are labeled by date and chemicals tested. The second folder includes data sheets from rainbow trout liver slice assays. This folder includes RNA concentrations and purities from all exposures, normalized RNA used for qPCR, plate layouts, raw data from qPCR runs, and preliminary figures from qPCR.
US EPA, ORD, GLTED Newton Creek, Superior, WI Studies, conducted fall 2017
In 2017 the Wisconsin DNR reported observation of a high incidence of fish with fin erosion in Newton Creek, which originates near the site of the Calumet (later Husky) oil refinery and flows approximately 2 miles through the city of Superior, WI, before discharging at the Hog Island Inlet of the Superior Bay. In fall 2017, the United States Environmental Protection Agency’s (US EPA) Office of Research and Development (ORD), Mid-Continent Ecology Division (now Great Lakes Toxicology and Ecology Division; GLTED) conducted several studies (two caged fish exposures and one resident fish collection) aimed at elucidating potential causes of the fin erosion phenotype reported. These studies were conducted in coordination and cooperation with both the Wisconsin DNR and Calumet/Husky refinery.
Bioactivity of the ubiquitous tire preservative 6PPD and degradant, 6PPD-quinone in fish and mammalian-based assays
6PPD-quinone (N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone), a transformation product of the antiozonant 6PPD (N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine) is a likely causative agent of coho salmon (Oncorhynchus kisutch) pre-spawn mortality. Stormwater runoff transports 6PPD-quinone into freshwater streams, rapidly leading to neurobehavioral and respiratory distress and rapid mortality in laboratory exposed coho salmon (LC50 41 – 95 ng/L) but causing no mortality in many (6 of 11) laboratory-tested species. Given this identified hazard, and potential for environmental exposure, we evaluated a set of U.S. Environmental Protection Agency’s high throughput assays for their capability to detect the large potency difference between 6PPD and 6PPD-quinone observed in coho salmon and screen for bioactivities of concern. Assays included transcriptomics in larval fathead minnow (FHM), developmental and behavioral toxicity in larval zebrafish, phenotypic profiling in a rainbow trout gill cell line, acute and developmental neurotoxicity in mammalian cells, and reporter transcription factor activity in HepG2 cells (Attagene). 6PPD was more consistently bioactive across tested assays, with distinct activity in the developmental neurotoxicity assay (mean 50th centile activity concentration =0.91 µM). 6PPD-quinone was less potent in FHM and zebrafish, and displayed minimal neurotoxic activity in mammalian cells, it was highly potent in altering organelle morphology in RTgill-W1 cells (phenotype altering concentration 0.024 µM [7.2 µg/L] compared to 0.96 µM [260 µg/L] for 6PPD). Although in vitro sensitivity of RTgill-W1 cells may not be as sensitive as Coho salmon exposed in vivo, the assay may be a promising approach to test chemicals for 6PPD-quinone-like activities. The other assays each identified unique bioactivities of 6PPD, with neurobehavioral and developmental neurotoxicity being most affected, indicating a need for further assessment of this chemical.
Eco-HTTr data analysis pipeline
This dataset provides the code-base for RNAseq data pipelining and analysis as part of the U.S. EPA, Great Lakes Toxicology and Ecology Division's ecological high throughput transcriptomics research program. The scripts are designed for use in either the U.S. EPA High Performance Computing (HPC) system or in R/R-studio. The scripts cover the entire workflow, starting with raw FASTQ files, to data preprocessing and quality control to differential gene expression analysis, benchmark dose modeling, and functional enrichment analysis. The code is accompanied by a protocol designed to be accessible to researchers with no prior experience in coding or working within a Linux-based computing environment. It aims to provide detailed, step-by-step instructions that will enable users to navigate through the various stages of the analysis pipeline effectively.
Cross-species sequence variability in host interferon antiviral pathway proteins and SARS-CoV-2 susceptibility
Cell entry and primary translation of genomic RNA are key events in the infection process for severe acute respiratory coronavirus 2 (SARS-CoV-2) to evade host innate immunity. Viral non-structural proteins (NSPs), structural, and accessory proteins suppress the interferon-I (IFN-I) antiviral response, leading to replication and spread of the COVID-19 disease. Zoonotic transmission has resulted in infections of more than 30 mammals. In previous studies the protein sequence conservation of the angiotensin converting enzyme 2 (ACE2) cell surface receptor and its binding affinity to the virus spike protein leading to cell entry have been evaluated. However, many species ranked as low susceptibility have become infected by the virus. In this study, the protein sequence conservation of 24 host protein targets was investigated including the entry proteins ACE2 and transmembrane serine protease 2 (TMPRSS2), 21 proteins in the IFN-I antiviral response pathway, and tethrin (bone marrow stromal antigen 2 [BST-2]), a protein that suppresses new virion release from cells. The Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool and other bioinformatics approaches were used to compare primary protein sequence similarity, conserved domains, and critical amino acids for the host-viral protein-protein interactions, based on published information. The results of this pathway approach suggest that variation in protein-protein interfaces is tolerated for many amino acid substitutions, and these substitutions follow taxonomic clades rather than correlating with empirically determined infected species. However, certain specific substitutions appear able to disrupt an interaction, with evidence that these may be specific to resistant species.
HPG axis model simulations from Morshead et al 2023 (https://doi.org/10.1016/j.aquatox.2023.106607)
Morshead ML, Jensen KM, Ankley GT, Vliet S, LaLone CA, Aller AV, Watanabe KH, Villeneuve DL. Putative adverse outcome pathway development based on physiological responses of female fathead minnows to model estrogen versus androgen receptor agonists. Aquat Toxicol. 2023 Aug;261:106607. doi: 10.1016/j.aquatox.2023.106607. Epub 2023 Jun 9. PMID: 37354817.
R code and data associated with Supplementary Figure 1. Comparison of model-based simulations (n=1000; summarized in box and whisker plot and black points) of Experiment 1 using a computational model of the fathead minnow hypothalamic-pituitary-gonadal axis developed by Li et al. (2011) with empirical results (red triangles).
Examining Effects of a Novel Estrogenic Perfluoro-alcohol, 1H,1H,8H,8H-Perfluorooctane-1,8-diol (FC8-diol), using the Fathead Minnow EcoToxChip
The Genome Canada funded EcoToxChip project has piloted development of a real-time PCR array format as a tool to support greater use of new approach methodologies in ecotoxicity testing. The present study applied the novel EcoToxChip technology to evaluate the expression of over 350 toxicologically-relevant gene targets in adult male fathead minnows that had been exposed to the estrogenic polyfluoroalkyl substance FC8-diol. The attached data files provide the raw data from the EcoToxChip platform, formatted for use with the EcoToxXplorer.ca and associated sample meta-data.
ROAR 2701 Laboratory OPs_GLTED
U.S. EPA, GLTED laboratory operating procedures associated with ROAR project 2701. Note, all procedures are for research and development purposes and subject to change based on performance over the course of the research. Major updates will be provided as new versions.
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| Datasets | 13 |
| Files: | 75 |
| Bytes: | 382.7 MB |
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