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Overview of Trajectory Analysis
Cells undergo changes to transition from one state to another as part of development, disease, and throughout life. Since those changes can be gradual, trajectory analysis attempts to describe progress through a biological process as a position along a path. Because biological processes are often complex, trajectory analysis builds branching trajectories where different paths can be chosen at different points along the trajectory. The progress of a cell along a trajectory from the starting point or root, can be quantified as a numeric value, pseudotime.
In Partek Flow, we use tools from Monocle 3 (1) to build trajectories, identify states and branch points, and calculate pseudotime values. The output of Trajectory analysis task includes an interactive 2D/3D visualization for viewing the trajectory trees and setting the root states (starting points of the trajectories). From the Trajectory analysis report, you can run a second task, Calculate pseudotime, which adds a numeric cell-level attribute, Pseudotime, calculated using the chosen root states.
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SubtitleText | Specifying Monocle 3 default options. If log normalization has been performed in Partek Flow, the Data is logged option will be selected automatically |
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AnchorName | monocle3 setup |
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Under the hood, Monocle 3 will perform log2 transformation of the gene count matrix (if Data is logged was unselected), scale the matrix (if Scaling was selected), and project the gene count matrix into the top 50 principal components. Next, the dimensionality reduction will be implemented by UMAP (using default settings of the reduce _ dimension command command).
Trajectory Analysis Result
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SubtitleText | Cell trajectory graph. Blue dots are individual cells (total count is displayed in the upper right). Black line represents the structure of the trajectory graph. Gray circles are nodes or leaves. The axes represent UMAP coordinates. |
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AnchorName | 3D trajectory plot |
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To show / hide cell trajectory tree and trajectory nodes, use the Extra data option on the Content card (Figure 2).
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SubtitleText | Extra data card enables the user to turn the trajectory tree and the trajectory nodes on or off |
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AnchorName | trajectory tree node |
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Pseudotime Analysis
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SubtitleText | Identification of root nodes for pseudotime analysis. The selected nodes are in dark gray |
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AnchorName | trajectory tree root node |
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Once you have identified all the root nodes, push the Calculate pseudotime button in the Selection panel (Figure 5).
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SubtitleText | Once the root cell nodes are selected, use the Calculate pseudotime button to start the calculation. In this example, seven root nodes were specified |
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AnchorName | calculate pseudotime |
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As a result, the cells will be annotated by pseudotime, using green to red gradient (start and end, respectively) (Figure 6). If, for a particular tree, no root node has been identified, those cells will be omitted from the pseudotime calculation and will be colored in black (Figure 9).
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SubtitleText | Cells annotated by pseudotime, from start (green) to end (red) |
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AnchorName | pseuodtime analysis |
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Following pseudotime calculation, three types of cell nodes will be annotated on the plot.
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SubtitleText | Following pseudotime analysis, three types of nodes can be identified on the trajectory plot. White circles - root nodes (beginning of pseudotime), black circles - branch nodes (splitting of differentiation pathway), and light gray nodes - leaves (outcome of differentiation pathway) |
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AnchorName | trajectory pseudotime node |
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Automatic Selection of the Root Node
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SubtitleText | When the option Programmatically select the default root node is turned on, the user can specify the cell attribute which specifies the root node information (Attribute for root nodes) and the level of that attribute corresponding to the earliest pseudotime (Attribute value for root nodes) |
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AnchorName | monoce3 setup root node |
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Once the options have been set, Monocle 3 will first group the cells according to which trajectory node they are nearest to. It then calculates the fraction of the cells from the earliest time point at each trajectory node. Finally, it picks the node with the highest prevalence of the early cells and treats it as the root node.
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SubtitleText | Trajectory tree colored by pseudotime, following automatic selection of the root node (2D view). White circles - root node (beginning of pseudotime), black circles - branch nodes (splitting of differentiation pathway), and light gray nodes - leaves (outcome of differentiation pathway), black dots - cells not connected to the main trajectory tree. |
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AnchorName | root node pseudotime |
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Difference Between Monocle 3 and Monocle 2
Major updates in Monocle 3 (compared to Monocle 2) include:
- Monocle 3 learns the principal trajectory graph in the UMAP space;
- the principal graph is smoothened and small branches are excluded;
- support for principal graphs with loops and convergence points;
- support for multiple root nodes.
References
- Cao J, Spielmann M, Qiu X, Huang X, Ibrahim DM, Hill AJ, Zhang F, Mundlos S, Christiansen L, Steemers FJ, Trapnell C, Shendure J. The single-cell transcriptional landscape of mammalian organogenesis. Nature. 2019 Feb;566(7745):496-502. doi: 10.1038/s41586-019-0969-x. Epub 2019 Feb 20. PMID: 30787437; PMCID: PMC6434952.
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