High-Throughput Transcriptomics Platform for Screening Environmental Chemicals

New approach methodologies (NAMs) that efficiently provide information about chemical hazard without using whole animals are needed to accelerate the pace of chemical safety assessments. Technological advancements in gene expression assays have made in vitro high-throughput transcriptomics (HTTr) a feasible option for NAMs-based hazard characterization of environmental chemicals. In the present study, we evaluated the Templated Oligo with Sequencing Readout (TempO-Seq) assay for HTTr concentration-response screening of a small set of chemicals in the human-derived MCF7 cell model. Our experimental design included a variety of reference samples and reference chemical treatments in order to objectively evaluate TempO-Seq assay performance. To facilitate analysis of these data, we developed a robust and scalable bioinformatics pipeline using open-source tools. We also developed a novel gene expression signature-based concentration-response modeling approach and compared the results to a previously implemented workflow for concentration-response analysis of transcriptomics data using BMDExpress. Analysis of reference samples and reference chemical treatments demonstrated highly reproducible differential gene expression signatures. In addition, we found that aggregating signals from individual genes into gene signatures prior to concentration-response modeling yielded in vitro transcriptional biological pathway altering concentrations (BPACs) that were closely aligned with previous ToxCast high-throughput screening (HTS) assays. Often these identified signatures were associated with the known molecular target of the chemicals in our test set as the most sensitive components of the overall transcriptional response. This work has resulted in a novel and scalable in vitro HTTr workflow that is suitable for high throughput hazard evaluation of environmental chemicals. Overall design: We evaluated the Templated Oligo with Sequencing Readout (TempO-Seq) assay for High-throughput transcriptomic screening of a small set of environmental chemicals. This assay yields sequencing reads of exactly 50 base pairs that can be rapidly aligned to generate gene counts, and is compatible with cell lysates prepared in multiwell format. The version of the TempO-Seq assay we evaluated provides nearly whole transcriptome coverage (>20,000 genes). This study encompasses 3 replicates of a 384-well plate design (1,134). The majority of wells contain MCF7 cells exposed to 44 test chemicals at 8 different concentrations (single replicate per test chemical, concentration, and plate), and additional reference samples and controls. Controls include DMSO vehicle treatments (4 per plate) and untreated wells (2 per plate). Reference samples include bulk lysate MCF7 samples (2 DMSO treated and 2 TSA treated samples per plate), reference chemical treatments (3 chemicals at single conc each, per plate), and vendor-provided reference RNA mixtures (UHRR and HBRR).