---
title: "Trajectory Analysis using Monocle3"
author: "Alex Germanos, Sonali Arora"
date: "Feb 24, 2022"
output: 
  html_document:
    toc: true
    theme: united
---

## Introduction

In this vignette, we perform a trajectory analaysis using Monocle3.
First we need to create a "new_cell_data_set" object from our exsiting Seurat object. 

```{r}

library(monocle3)
library(Seurat)
library(SeuratWrappers)

seurat.epi = readRDS("seurat_cleaned.epi.rds")
my.cds =as.cell_data_set(seurat.epi)

```

Next we need to perform the trajectory analysis using monocle3

```{r}
# do the trajectory analysis : 
my.cds <- learn_graph(my.cds, use_partition = TRUE)
t1 = plot_cells(my.cds, label_groups_by_cluster = FALSE, label_leaves = FALSE, label_branch_points = FALSE)  + 
  ggtitle("Initial Trajectory Plot  ") 

# find root node to start trajectory :
cell_ids <- which(colData(cds)[, "seurat_clusters"] == 4)
closest_vertex <- my.cds@principal_graph_aux[["UMAP"]]$pr_graph_cell_proj_closest_vertex
closest_vertex <- as.matrix(closest_vertex[colnames(my.cds), ])
root_pr_nodes <-
 igraph::V(principal_graph(my.cds)[["UMAP"]])$name[as.numeric(names(which.max(table(closest_vertex[cell_ids, ]))))]
my.cds <- order_cells(my.cds, root_pr_nodes=root_pr_nodes)


# plot to visualize pseudotime
t2 = plot_cells(my.cds,
               color_cells_by = "pseudotime", label_cell_groups=FALSE,
               label_leaves=FALSE,   label_branch_points=FALSE,
               graph_label_size=1.5,  cell_size =0.5) + ggtitle(" Psuedotime for Epithelial Cells")

```

Finally, we can add the trajectory data to the seurat object and save it 

```{r}

seurat.epi <- AddMetaData(
  object = seurat.epi,
  metadata = my.cds@principal_graph_aux@listData$UMAP$pseudotime,
  col.name = "Epithelial.Psuedotime"
)

# check if plot is made correctly with Seurat object
FeaturePlot(seurat.epi, c("Epithelial.Psuedotime"), pt.size = 0.1) & scale_color_viridis_c()

# save objects for future use.
saveRDS(my.cds, "monocle3_cds.epi.rds")
saveRDS(seurat.epi, "sueurat_cleaned.epi.with.monocle3.rds")

```