catcheR_pseudotime - Pseudotime
==================================

Since Monocle pseudotime calculation requires interactive root cell selection, the first step must be performed manually within ``monocle3``.

Step-by-step
------------

#. Load ``processed_cds.RData`` in R, then calculate pseudotime and plot it:

   .. code-block:: r

      library(monocle3)
      cds <- order_cells(cds)
      pt = as.data.frame(pseudotime(cds))
      names(pt) = c("pseudotime")
      write.csv(pt, paste0(dir, "/pseudotime.csv"))

      plot_cells(cds,
                 color_cells_by = "pseudotime",
                 label_cell_groups = FALSE,
                 label_leaves = FALSE,
                 label_branch_points = FALSE,
                 graph_label_size = 1.5)

#. Your working folder should now contain:

   - Outputs from ``catcheR_load``
   - The ``pseudotime.csv`` file created above

#. Run ``catcheR_pseudotime``:

   .. code-block:: r

      catcheR_pseudotime(
          group = c("docker", "sudo"),
          folder, 
          cds,
          pseudotime, 
          all = FALSE
      )

**Example usage:**

.. code-block:: r

   catcheR_pseudotime(
       group = "docker",
       folder = "/path/to/working/folder/", 
       cds = "processed_cds.RData",
       pseudotime = "pseudotime.csv"
   )

The ``all`` argument is a logical value:

- ``FALSE`` (default): compare target vs. all controls together  
- ``TRUE``: compare target vs. each control separately

Outputs
-------

The following files are generated by ``catcheR_pseudotime``:

#. **cumulative_frequency_pseudotime.pdf**  
   - Plots showing the number of cells at each pseudotime point, grouped by gene, shRNA, and clone.
   
   .. image:: cumulative_frequency_pseudotime_gene.pdf
   
   .. image:: cumulative_frequency_pseudotime_shRNA.pdf
   
   .. image:: cumulative_frequency_pseudotime_clone.pdf

#. **ks_statistics_*.csv**  
   - Kolmogorov-Smirnov test results comparing cumulative frequencies along pseudotime between knockdown and controls.

#. **Volcano plots**  
   - Visualize significance and effect size (fold change) of the Kolmogorov-Smirnov test, across different comparisons.
   
   .. image:: volcano_ks_stats_vs_control_genes_allFALSE.pdf
   
   .. image:: volcano_shRNA_ks_statistics_vscontrol_genes_allFALSE.pdf

#. **correlated_pseudotime_gene_exp.pdf**  
   - UMAP plot highlighting genes whose expression is most correlated with pseudotime.
   
   .. image:: correlated_pseudotime_gene_expX12.pdf