TockyLocus

TockyLocus standardizes quantitative analysis of flow cytometric fluorescent timer data by implementing a five-category scheme based on biologically grounded angular intervals to categorize cells into Tocky Loci and enable downstream statistical testing and visualization of transcriptional dynamics.

Key Features:

• Five-locus categorization: Categorizes cells into five biologically relevant loci based on Timer Angle values using angular intervals anchored at 0 degrees, 45 degrees, and 90 degrees.
• Timer Angle analysis: Optimizes quantitative analysis methods for Timer Angle, the key parameter within the Tocky framework, using computational modelling of Timer protein kinetics.
• Statistical testing: Enables downstream statistical testing of transcriptional dynamics in flow cytometry data.
• Visualization: Provides methods for visualization of transcriptional dynamics data.
• Robustness validation: Validated using simulated and experimental datasets, including comprehensive simulations with synthetic spike-in datasets and real-world datasets from Nr4a3-Tocky and Foxp3-Tocky mice.
• Reproducible interpretation: Standardizes quantitative analysis without reliance on arbitrary gating or complex assumptions.

Scientific Applications:

• T cell activation studies: Investigates dynamic cellular processes such as T cell activation using the Timer-of-Cell-Kinetics-and-Activity (Tocky) framework.
• Transcriptional dynamics analysis: Links underlying biological dynamics to the percentage of cells in each Tocky Locus for investigating transcriptional dynamics in immunology and related fields.
• Nr4a3-Tocky mouse studies: Analyzes transcriptional dynamics in Nr4a3-Tocky reporter mouse datasets.
• Foxp3-Tocky mouse studies: Analyzes transcriptional dynamics in Foxp3-Tocky reporter mouse datasets.

Methodology:

TockyLocus builds on the TockyPrep preprocessing framework and implements optimized algorithms for Timer Angle analysis. Computational modelling of Timer protein kinetics defines transcriptional dynamics in relation to key anchoring points in Timer Angle values. The five-locus model was systematically evaluated against categorization schemes ranging from three to seven loci on real-world datasets.