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

Paradigms and Technologies


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.


Hsieh, Jui-Hua; Sedykh, Alexander; Huang, Ruili; Xia, Menghang; Tice, Raymond R;


  • Artifacts
  • Data Interpretation, Statistical
  • Drug Discovery/ methods
  • Drug Discovery/ standards
  • High-Throughput Screening Assays/ methods
  • High-Throughput Screening Assays/ standards
  • Humans
  • Reproducibility of Results
  • Signal-To-Noise Ratio
  • Small Molecule Libraries
  • United States

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