Eco-HTTr
This space houses datasets associated with ecological high throughput transcriptomics research
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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.
Transcriptomics-based points of departure for Daphnia magna exposed to 18 per- and polyfluoroalkyl substances.
Daphnia magna were exposed to multiple concentrations of 22 different PFAS for 24 h, in 96-well plate format. Following exposure, whole body RNA was extracted and extracts, each representing five exposed individuals, were subjected to RNA sequencing. Following analytical measurements to verify PFAS exposure concentrations, and quality control on processed cDNA libraries for sequencing, concentration-response modeling was applied to the data sets for 18 of the tested compounds, and the concentration at which a concerted molecular response occurred (transcriptomic point of departure; tPOD) was calculated. This file provides the processed and normalized count matrices for each of the 18 PFAS evaluated along with exported results from BMDExpress 3.0 (tab delimited .txt files).
Raw data were submitted to the National Center for Biotechnology Information Sequence Read Archive (Bioproject PRJNA1018977) and Gene Expression Omnibus (GEO Accession GSE245268). Sample meta data copied here.
Zebrafish embryo/larval toxicity testing with PFAS to address uncertainties in transcriptomic point of departure estimates across species.
Background:
Mounting evidence suggests that chemical concentrations that do not elicit concerted molecular responses over relatively short exposure durations (e.g., 24 h to 5 d) generally do not elicit adverse effects, even over much longer exposure durations. This has led to proposals to implement an omics-based regulatory testing paradigm which would use transcriptomic points of departure (tPOD; a benchmark dose/concentration -based treatment level below which a concerted gene expression response is not observed) as a health protective exposure level for risk assessment (Johnson et al. 2022). While initial research on the application of tPODs has focused on human health protection, more recently our research team and others have started to explore application of this concept for ecosystem protection as well. To evaluate the scientific underpinnings of the approach, two critical questions as it pertains to regulatory application are:
1) How variable are tPODs as a function of common experimental design variables?
2) How do tPODs for the same chemical compare across species?
To examine these questions, zebrafish (Danio rerio) embryos were exposed to three different per- or polyfluoroalkyl substances (PFAS), perfluorooctanesulfonic acid (PFOS; DTXSID3031864); perfluorooctanoic acid (PFOA; DTXSID8031865); and perfluorohexane sulfonate (PFHxS; DTXSID90892476) in concentration-response, and then whole-body gene expression was determined using RNA sequencing (RNAseq). Embryos were exposed to each of the three PFAS, as well as a chlorpyrifos positive control, over seven distinct exposure periods (6 hours post fertilization [hpf]-120 hpf; 6 hpf-48 hpf; 6 hpf-24 hpf; 24-48 hpf; 24 hpf – 120 hpf; 48 hpf-120 hpf; and 96 hpf-120 hpf; Figure 1) with the goal of determining how much the tPOD varies as a function of the developmental window over which the organisms were exposed. In a separate study (CSS.1.7.5), tPODs for PFOS, PFOA, and PFHxS were generated for several other species including fathead minnow (Pimephales promelas), Daphnia magna (a crustacean), Chironomous dilutus (an insect) and Raphidocelis subcapitata (an algae). Thus, the tPODs generated for zebrafish embryos can also be compared against these data.
Dataset:
Processed RNAseq data are provided as normalized count matrices (counts per million reads; filtered to remove genes with <15 reads across all samples; log2 transformed with a pseudo count of 1 added to avoid negative value) with individual genes as rows and samples as columns. Data sets are organized into 21 count matrices, one for each exposure window evaluated for each chemical (T1-T7; Table 1). A meta-data file that that defines the exposure window, test chemical, treatment concentration, and replicate number for each sample is also provided. RNAseq was repeated for a sub-set of samples. Repeated samples are indicated in the metadata file.
AOP-Wiki xml for derivation and evaluation of cross-species AOP network for thyroid hormone system disruption
Thyroid hormone system disrupting compounds are considered potential threats for human and environmental health. Multiple adverse outcome pathways (AOPs) for thyroid hormone system disruption (THSD) are being developed in different taxa. Combining these AOPs results in a cross-species AOP network for THSD which may provide an evidence-based foundation for extrapolating THSD data across vertebrate species and bridging the gap between human and environmental health. This review aimed to advance the description of the taxonomic domain of applicability (tDOA) in the network to improve its utility for cross-species extrapolation. We focused on the molecular initiating events (MIEs) and adverse outcomes (AOs) and evaluated both their plausible domain of applicability (taxa they are likely applicable to) and empirical domain of applicability (where evidence for applicability to various taxa exists) in a THSD context.
The evaluation showed that all MIEs in the AOP network are applicable to mammals. With some exceptions, there was evidence of structural conservation across vertebrate taxa and especially for fish and amphibians, and to a lesser extent for birds, empirical evidence was found. Current evidence supports the applicability of impaired neurodevelopment, neurosensory development (e.g., vision) and reproduction across vertebrate taxa. The results of this tDOA evaluation are summarized in a conceptual AOP network that helps prioritize (parts of) AOPs for a more detailed evaluation. In conclusion, this review advances the tDOA description of an existing THSD AOP network and serves as a catalog summarizing plausible and empirical evidence on which future cross-species AOP development and tDOA assessment could build.
Pilot testing and optimization of a larval fathead minnow high throughput transcriptomics assay
Concentrations at which global gene expression profiles in cells or animals exposed to a test substance start to differ significantly from those of controls have been proposed as an alternative point of departure for use in screening level hazard assessment. The present study describes pilot testing of a high throughput compatible transcriptomics assay with larval fathead minnows. One day post hatch fathead minnows were exposed to eleven different concentrations of three metals, three selective serotonin reuptake inhibitors, and four neonicotinoid-like compounds for 24 h and concentration response modeling was applied to whole body gene expression data. Transcriptomics-based points of departure (tPODs) were consistently lower than effect concentrations reported in apical endpoint studies in fish. However, larval fathead minnow-based tPODs were not always lower than concentrations reported to elicit apical toxicity in other aquatic organisms like crustaceans or insects. Random in silico subsampling of data from the pilot assays was used to evaluate various assay design and acceptance considerations such as transcriptome coverage, number of replicate individuals to sequence per treatment, and minimum number of differentially expressed genes to produce a reliable tPOD estimate. Results showed a strong association between the total number of genes for which a concentration response relationship could be derived and the overall variability in the resulting tPOD estimates. We conclude that tPODs based on fewer than 15 differentially expressed genes are likely to be unreliable for screening and that interindividual variability in gene expression profiles appears to be a more significant driver of tPOD variability than sample size alone. Results represent initial steps toward developing high throughput transcriptomics assays for use in ecological hazard screening.
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