Setting up Trajectory Analysis

To run Trajectory analysis tool, select the Normalized counts data node (or equivalent) and go to the toolbox: Exploratory analysis > Trajectory analysis

There configuration dialog presents two options.

1. Dimensionality of reduced space. This option specifies the number of UMAP dimensions that the original data are reduced to, in order to learn the trajectory tree (dimensionality of original data equals the number of genes). Default is two, meaning that the trajectory plot will be draw in two dimensions. To get a 3D trajectory plot, increase this option to 3.
2. Scaling. Normalized expression values can be further transformed by scaling to unit variance and zero mean (i.e. converting to Z score). The use of this option is recommended. 

Trajectory Analysis Result

Result of running Trajectory analysis in Partek Flow is the Trajectory result data node. Double clicking on the node opens a Data Viewer window with the trajectory plot (Figure xxx). Cell trajectory graph shows position of each cell (blue dot) with respect to the UMAP coordinates (axes). Cell trajectories (one or more, depending on the data set) are depicted as black lines. Gray circles are trajectory nodes (i.e. cell communities).

To show / hide cell trajectory tree and trajectory nodes, use the Extra data option on the Content card (Figure xxx).

Pseudotime Analysis

To perform pseudotime analysis, you need to point to the cells at the beginning of the biological process you are interested in. For example, cells at the earliest stage of differentiation sequence. To start select the root cell nodes (gray circles) by left-click. If the trajectory result consists of more than one trajectory tree, you can specify more than one root node, e.g. one root node per trajectory tree (ctrl & click). If no root node is specified for a tree, that tree will not be included in the pseudotime calculation. Figure xxx shows an example where seven root nodes were identified.

Once you have identified all the root nodes, push the Calculate pseudotime button in the Selection panel (Figure xxx).

As a result, the cells will be annotated by pseudotime, using green to red gradient (start and end, respectively) (Figure xxx). If, for a particular tree, no root node has been specified, those cells will be omitted from the pseudotime calculation and will be colored in gray (not shown).

As a result of pseudotime calculation, three types of cell nodes will become apparent on the plot.

Root node (white). Root nodes are start points of the pseudotime and were defined by the user in the previous step (e.g. node 7 in Figure xxx).

Branch node (black). Branch nodes indicate where the trajectory tree forks out; i.e. each branch represents a different cell fate or different trajectory (e.g. nodes 1 and 4 in Figure xxx).

Leaf (light gray). Leaves correspond to different cell fates / different trajectory outcomes (e.g. nodes 3, 8, and 11 in Figure xxx). The leaves correspond to cell states of Monocle 2.