TockyPrep: data preprocessing methods for flow cytometric fluorescent timer analysis
File(s)
Published version
Author(s)
Ono, Masahiro
Type
Journal Article
Abstract
Background:
Fluorescent Timer proteins, which display time-dependent changes in their emission spectra, are invaluable for analyzing the temporal dynamics of cellular events at the single-cell level. We previously developed the Timer-of-cell-kinetics-and-activity (Tocky) tools, utilizing a specific Timer protein, Fast-FT, to monitor temporal changes in cellular activities. Despite their potential, the analysis of Timer fluorescence in flow cytometry is frequently compromised by variability in instrument settings and the absence of standardized preprocessing methods. The development and implementation of effective data preprocessing methods remain to be achieved.
Results:
In this study, we introduce the R package that automates the data preprocessing of Timer fluorescence data from flow cytometry experiments for quantitative analysis at single-cell level. Our aim is to standardize Timer data analysis to enhance reproducibility and accuracy across different experimental setups. The package includes a trigonometric transformation method to elucidate the dynamics of Fluorescent Timer proteins. We have identified the normalization of immature and mature Timer fluorescence data as essential for robust analysis, clarifying how this normalization affects the analysis of Timer maturation. These preprocessing methods are all encapsulated within the TockyPrep package.
Conclusions:
TockyPrep is available for distribution via GitHub at https://github.com/MonoTockyLab/TockyPrep, providing tools for data preprocessing and basic visualization of Timer fluorescence data. This toolkit is expected to enhance the utility of experimental systems utilizing Fluorescent Timer proteins, including the Tocky tools.
Fluorescent Timer proteins, which display time-dependent changes in their emission spectra, are invaluable for analyzing the temporal dynamics of cellular events at the single-cell level. We previously developed the Timer-of-cell-kinetics-and-activity (Tocky) tools, utilizing a specific Timer protein, Fast-FT, to monitor temporal changes in cellular activities. Despite their potential, the analysis of Timer fluorescence in flow cytometry is frequently compromised by variability in instrument settings and the absence of standardized preprocessing methods. The development and implementation of effective data preprocessing methods remain to be achieved.
Results:
In this study, we introduce the R package that automates the data preprocessing of Timer fluorescence data from flow cytometry experiments for quantitative analysis at single-cell level. Our aim is to standardize Timer data analysis to enhance reproducibility and accuracy across different experimental setups. The package includes a trigonometric transformation method to elucidate the dynamics of Fluorescent Timer proteins. We have identified the normalization of immature and mature Timer fluorescence data as essential for robust analysis, clarifying how this normalization affects the analysis of Timer maturation. These preprocessing methods are all encapsulated within the TockyPrep package.
Conclusions:
TockyPrep is available for distribution via GitHub at https://github.com/MonoTockyLab/TockyPrep, providing tools for data preprocessing and basic visualization of Timer fluorescence data. This toolkit is expected to enhance the utility of experimental systems utilizing Fluorescent Timer proteins, including the Tocky tools.
Date Issued
2025-02-08
Date Acceptance
2025-01-20
Citation
BMC Bioinformatics, 2025, 26
ISSN
1471-2105
Publisher
BMC
Journal / Book Title
BMC Bioinformatics
Volume
26
Copyright Statement
© The Author(s) 2025. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
License URL
ttp://creativecommons.org/licenses/by/4.0/
Identifier
10.1186/s12859-025-06058-8
Subjects
Fluorescent Timer Protein
Tocky
Flow Cytometry
Data preprocessing
Nr4a3-Tocky
Publication Status
Published
Article Number
ARTN 44