Figure 5 - Pseudotime Analysis

Author

Ashok Patowary

library(monocle3) # requires Bioconductor version 3.14 https://cole-trapnell-lab.github.io/monocle3/docs/installation/
library(Seurat)
#library(SeuratData)
library(SeuratWrappers) # remotes::install_github('satijalab/seurat-wrappers@9652bdd') (for Seurat >= 3.0.1; use latest release for Seurat >= 4.2.0)
library(ggplot2)
library(patchwork)
library(magrittr)

isoT4k_filter = readRDS("data/working/isoT4k_filter.rds")
levels(isoT4k_filter$orig.ident) = gsub("\\.", "-", levels(isoT4k_filter$orig.ident)) # ExM.U becomes ExM-U
isoT4k_filter

cds <- as.cell_data_set(isoT4k_filter)
cds <- cluster_cells(cds,reduction_method = "UMAP")
cds <- learn_graph(cds, use_partition = FALSE)

##  Order cells
cds <- order_cells(cds, reduction_method = "UMAP")

cds.sub <- subset(as.Seurat(cds, assay = NULL), monocle3_partitions == 1)
cds.sub <- as.cell_data_set(cds.sub)
cds.sub <- learn_graph(cds.sub)
#vRG = row.names(isoT4k_filter@meta.data %>% filter(isoT4k_filter@meta.data$isoCellType == "vRG"))
#cds <- order_cells(cds, reduction_method = "UMAP", root_cells = vRG)

cds.sub <- order_cells(cds.sub, reduction_method = "UMAP")

plot_cells(cds.sub, color_cells_by = "pseudotime", label_cell_groups = FALSE, label_leaves = FALSE, 
           label_branch_points = FALSE, group_cells_by = "cluster")

#ggsave("~/Desktop/Plots_N/upload/pseudotime.ExN", device ="pdf", dpi=600, width = 6, height = 5, units = "in")
ggsave("output/figures/Fig5/Fig5_pseudotime.pdf", width = 4, height = 10/3)