Cells that differentiate display a continuous spectrum of states

• Transcriptional program for activation and differentiation

Individual cells will differentiate in an unsynchronized manner

• Each cell is a snapshot of differentiation time

Pseudotime – abstract unit of progress

• Distance between a cell and the start of the trajectory

Are you sure that you have a developmental trajectory?

Do you have intermediate states?

Do you believe that you have branching in your trajectory?

Be aware, any dataset can be forced into a trajectory without any biological meaning!

First make sure that gene set and dimensionality reduction captures what you expect.

It is a NON-LINEAR method of dimensionality reduction.

The distances between points are measured as probability from going from one to another.

The data must present connectivity (transitional cells).

In reality, distance in multidimensional space reflects difference in transcriptional landscape, not actual time.

Necessary to have a continuum of states among your cells (Will not work well with 2 distinct clusters)

May work with single time-point if ongoing differentiation process (It is better to have multiple experimental time points)

Be aware, any dataset can be forced into a trajectory without any biological meaning!