A Data Analysis Pipeline Accounting for Artifacts in Tox21 Quantitative High-Throughput Screening Assays

Abstract

A main goal of the U.S. Tox21 program is to profile a 10K-compound library for activity against a panel of stress-related and nuclear receptor signaling pathway assays using a quantitative high-throughput screening (qHTS) approach. However, assay artifacts, including nonreproducible signals and assay interference (e.g., autofluorescence), complicate compound activity interpretation. To address these issues, we have developed a data analysis pipeline that includes an updated signal noise-filtering/curation protocol and an assay interference flagging system. To better characterize various types of signals, we adopted a weighted version of the area under the curve (wAUC) to quantify the amount of activity across the tested concentration range in combination with the assay-dependent point-of-departure (POD) concentration. Based on the 32 Tox21 qHTS assays analyzed, we demonstrate that signal profiling using wAUC affords the best reproducibility (Pearson's r = 0.91) in comparison with the POD (0.82) only or the AC(50) (i.e., half-maximal activity concentration, 0.81). Among the activity artifacts characterized, cytotoxicity is the major confounding factor; on average, about 8% of Tox21 compounds are affected, whereas autofluorescence affects less than 0.5%. To facilitate data evaluation, we implemented two graphical user interface applications, allowing users to rapidly evaluate the in vitro activity of Tox21 compounds.

Keywords: Tox21; concentration-response curve; in vitro activity profiling; qHTS data analysis.

Figure 1

Quantitative high-throughput screening profiling pipeline. (a) Blue components represent the analysis at plate level, magenta components represent the analysis at the source level, and the green component represents the analysis at compound level. (b) Compound hit calling/ranking schema.

Figure 2

Assay reproducibility by weighted version of the area under the curve (wAUC). (a) Comparison of cytotoxicity assays based on three signal-processing protocols as a function of Curvep baseline noise threshold (THR). The solid/dashed line represents the median/25th percentile of 27 Pearson's r values (3 agencies × 3 compound plates × 3 screening plates). (b) Reproducibility within compound plates presented as highest Pearson's r for three corresponding screening-plate comparisons. Note: NTP_C plate in TR activation-type assay is removed (Pearson's r = 0.35) to give higher resolution to the plot.

Figure 3

The signal reproducibility between nine plates and three batches across 32 assays using different activity metrics (weighted version of the area under the curve (wAUC), point of departure (POD), and AC₅₀) is presented as box-and-whisker plots. Each box-and-whisker-plot is constructed based on 32 median Pearson's r values from pairwise plate comparisons of each assay. The outliers shown in the plots are all viability assays: mitochondria membrane potential (high-quality AC₅₀), glucocorticoid receptor antagonism (medium-quality AC₅₀), and androgen receptor (full-length) antagonism (POD).