Author: Dr Masahiro Ono
Date: 13 March 2026
Introduction
Experimentally resolving temporal transcriptional dynamics at single-cell resolution in vivo is a major challenge, as standard scRNA-seq only provides cross-sectional “snapshots”. CanonicalTockySeq bridges this gap by integrating a molecular clock of T-cell receptor (TCR) signalling—based on the Nr4a3-Tocky Fluorescent Timer—with scRNA-seq to establish an experimentally anchored temporal reference.
By using landmark Tocky fractions (New, Persistent, and Arrested) as biological ground truths, the package constructs a transcriptomic manifold in canonical space with unique conical geometry. This allows for the decoupling of temporal progression (geodesic angle) from signalling strength (radial intensity).
Key Features
The CanonicalTockySeq framework provides a robust methodology for capturing continuous, endogenous temporal resolution anchored to biological history:
CanonicalTockySeq (RDA Architecture)
Implements a supervised Canonical Redundancy Analysis (RDA) designed for single-cell Tocky data.
Constrains high-dimensional transcriptomic space using reference Tocky signatures to derive a model-driven path of differentiation.
Projects individual single cells onto this structure to visualize their state while preserving biological heterogeneity.
GradientTockySeq
Utilizes Piecewise Spherical Linear Interpolation (SLERP) to connect landmark biplot vectors (New → Persistent → Arrested).
Generates a curved, geodesic path along the transcriptomic hypersphere, preventing artifacts inherent to standard polynomial trajectories.
Maps cells to a 0–90° Tocky Time coordinate (angle) and a normalized Tocky Intensity (magnitude).
Availability
- CanonicalTockySeq is available at GitHub: CanonicalTockySeq.
Citation
If you use CanonicalTockySeq in your research, please cite the relevant publication and the R package.
You can cite the specific release of this software via its Zenodo DOI:
bioRxiv preprint
For a detailed description of the biological applications and the underlying methodology of CanonicalTockySeq, please refer to our preprint:
Temporal Mechanisms of T-Cell Fate Decisions under Immune Checkpoint Blockade Resolved by CanonicalTockySeq. Jehanne Hassan, Omnia Reda, Nobuko Irie, Malin Pedersen, Shane Foo, Lizzie Appleton, Il-mi Okazaki, Taku Okazaki, Yorifumi Satou, Kevin Harrington, Alan Melcher, Masahiro Ono. bioRxiv (2026). DOI: 10.64898/2026.03.10.710825
📄 Read the preprint on bioRxiv
@article{Ono2026CanonicalTocky,
author = {Ono, Masahiro and others},
title = {Temporal Mechanisms of T-Cell Fate Decisions under Immune Checkpoint Blockade Resolved by CanonicalTockySeq},
elocation-id = {2026.03.10.710825},
year = {2026},
doi = {10.64898/2026.03.10.710825},
publisher = {Cold Spring Harbor Laboratory},
journal = {bioRxiv},
url = {https://www.biorxiv.org/content/10.64898/2026.03.10.710825v1}
}R package
@Manual{OnoCanonicalTockySeq,
title = {CanonicalTockySeq: Canonical Tocky Analysis for Temporal Gradient on a Transcriptomic Manifold},
author = {Masahiro Ono},
year = {2026},
note = {R package version 0.1.0},
url = {[https://github.com/MonoTockyLab/CanonicalTockySeq](https://github.com/MonoTockyLab/CanonicalTockySeq)},
doi = {10.5281/zenodo.19003235}
}License
CanonicalTockySeq is licensed under the Apache License 2.0. See the LICENSE file for details.
Copyright and intellectual property
Copyright © Masahiro Ono.
Original graphical content in this repository, including characters and logos, is protected by copyright unless otherwise stated.
A patent application relating to aspects of the methodology has been filed.
Contact
For questions about the package, please contact: m.ono@imperial.ac.uk
The Ono Lab (MonoTockyLab)
The Masahiro Ono Lab (MonoTockyLab) develops experimental and computational approaches to study immune cell dynamics, with a particular focus on the temporal regulation of gene expression in T cells.
The lab is known for the development of Tocky (Timer of cell kinetics and activity), a platform that uses Fluorescent Timer proteins to analyse transcriptional and signalling dynamics in vivo at single-cell resolution. Our research integrates mouse genetics, immunology, flow cytometry, single-cell omics, and computational modelling.
Current research directions include:
- cancer immunology and immunotherapy
- temporal mechanisms of T cell activation, differentiation, and tolerance
- Foxp3 transcriptional dynamics and their regulation in vivo
- computational methods for time-resolved single-cell analysis, including CanonicalTockySeq
Principal Investigator: Dr Masahiro Ono, Reader in Immunology at Imperial College London.
Dr Ono is the creator of Tocky, spanning both its transgenic reporter systems and associated analytical frameworks.
Contact and More
Email: 
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Professional Homepage: Imperial College London - Masahiro Ono