...

In Partek Flow, we use tools from Monocle 3 [(1] to ) 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. 

Major updates in Monocle 3 (compared to Monocle 2) include:

...

Prerequisites for the Analysis

Trajectory analysis by Monocle 3 requires data normalisation and preprocessing. We suggest to run the Recommended normalization from the Normalization and scaling section of the toolbox (i.e. normalize by counts per million, add offset of 1, and then log2 transform). According to the Monocle 3 authors, you may want to filter in the top 5,000 genes with the highest variance (2,000 for datasets with fewer than 5,000 cells, and 300 for datasets with fewer than 1,000 cells) (1). Those number should be used as a guidance for the first-pass analysis and may need to be optimized, depending on the actual analysis.

Setting up Trajectory Analysis

...

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 (1). 

Under the hood, Monocle 3 will 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).

Trajectory Analysis Result

...

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

1. 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.

...