---
title: "Cluster Annotation (part 2)"
author: "Nina-Lydia Kazakou"
date: "18/06/2021"
output: html_document
---
# load libraries
```{r message=FALSE}
library(SingleCellExperiment)
library(Seurat)
library(scater)
library(scran)
library(devtools)
library(dplyr)
library(ggsci)
library(tidyverse)
library(Matrix)
library(scales)
library(here)
```
# Set the colour pallete
```{r}
mypal <- pal_npg("nrc", alpha = 0.7)(10)
mypal2 <-pal_tron("legacy", alpha = 0.7)(7)
mypal3 <- pal_lancet("lanonc", alpha = 0.7)(9)
mypal4 <- pal_simpsons(palette = c("springfield"), alpha = 0.7)(16)
mypal5 <- pal_rickandmorty(palette = c("schwifty"), alpha = 0.7)(6)
mypal6 <- pal_futurama(palette = c("planetexpress"), alpha = 0.7)(5)
mypal7 <- pal_startrek(palette = c("uniform"), alpha = 0.7)(5)
mycoloursP<- c(mypal, mypal2, mypal3, mypal4, mypal5, mypal6, mypal7)
show_col(mycoloursP, labels =F)
```
# Load normalised seu.object
```{r}
norm.co.seu <- readRDS(here("data", "norm.co.seu.rds"))
dim(norm.co.seu) # 22735 12923
```
```{r}
DefaultAssay(norm.co.seu) <- "RNA"
Idents(norm.co.seu) <- "RNA_snn_res.0.5"
```
```{r}
DimPlot(norm.co.seu, reduction = "umap", pt.size = 0.5, cols = mycoloursP, label = TRUE) & NoAxes()
```
I have now manually looked at the markers and here are my thoughts:
# I have devided the object into two subsets (neu: containing what I think is neuronal populations & gli: what I think contains glia cells;
Cluster8 & Cluster 11 looked a bit ambiguous, so they are included in both subsets)
```{r}
neu <- subset(norm.co.seu, ident = c(0, 1, 2, 3, 6, 7, 8, 9, 11)) #highlighted in purple
dim(neu) # 22735 10778
gli <- subset(norm.co.seu, ident = c(4, 5, 8, 10, 11, 12, 13)) #highlighted in red
dim(gli) # 22735 3074
```
1) There are no pericytes/endothelial cells in this dataset (could Cluster 8 contain pericytes?)
2) There are no microglia in this dataset
3) Oligodendroglia are located in Cluster 12, with some OLIG1+/OLIG2+ cells residing withing Cluster 4
```{r fig.width=7, fig.height=7, fig.fullwidth=TRUE}
FeaturePlot(gli, features = c("OLIG1", "OLIG2", "SOX10"), label = TRUE, cols = c("grey", "red")) & NoAxes()
```
```{r fig.width=7, fig.height=5, fig.fullwidth=TRUE}
VlnPlot(gli, features = c("OLIG1", "OLIG2", "SOX10"), cols = mycoloursP, ncol = 2, pt.size = 0.01)
```
4) Cluster 4 could be characterised as Radial Glia or Glial Progenitors; I believe it gives rise to Cluster 12 (oligodendroglia)
& Cluster 10 (expresses some mature astrocyte markers, but mostly immature, so it could be characterised as Intermediate Glial Progenitors); It does look like an astrocyte population too
```{r fig.width=7, fig.height=15, fig.fullwidth=TRUE}
FeaturePlot(gli, features = c("HOPX", "HES1", "HES5", "VIM", "PAX6", "PTPRZ1", "SPARCL1", "BCAN", "SLC1A3","GLI3"), label = TRUE, ncol = 2, cols = c("grey", "red")) &NoAxes()
```
```{r fig.width=7, fig.height=12, fig.fullwidth=TRUE}
VlnPlot(gli, features = c("HOPX", "HES1", "HES5", "VIM", "PAX6", "PTPRZ1", "SPARCL1", "BCAN", "SLC1A3","GLI3"), cols = mycoloursP, ncol = 2, pt.size = 0.01)
```
5) Cluster 13 is what I would call mature Astrocytes
```{r fig.width=7, fig.height=7, fig.fullwidth=TRUE}
FeaturePlot(gli, features = c("AQP4", "GFAP", "S100B", "SPON1"), label = TRUE, cols = c("grey", "red")) &NoAxes()
```
```{r fig.width=7, fig.height=5, fig.fullwidth=TRUE}
VlnPlot(gli, features = c("AQP4", "GFAP", "S100B", "SPON1"), cols = mycoloursP, ncol = 2, pt.size = 0.01)
```
6) Cluster 5 resembles Cluster 4 a lot, but I think it contains Radial Glia cells that only give rise to astrocytes
or it could also be characterised as astrocytes
7) Cluster 11 is positive for proliferation markers, but also expresses some early neuronal markers, so it could be called Cycling Progenitors
```{r fig.width=7, fig.height=3, fig.fullwidth=TRUE}
FeaturePlot(norm.co.seu, features = c("MKI67", "TOP2A"), label = TRUE) &NoAxes()
```
8) Cluster 8 could be characterised as Early Neurons, highlighting the transition from glia to neurons in the umap plot
This cluster is also positive for PDGFRB which usually characterises pericytes, but it also extensively expressed by
neurons,chorioid plexus, vascular smooth muscle cells, and pericytes of the human brain, particularly the basal
ganglia and the dentate nucleus
```{r}
FeaturePlot(neu, features = "STMN2", label = TRUE) & NoAxes() #early neuronal marker
```
```{r}
FeaturePlot(norm.co.seu, features = "PDGFRB", label = TRUE) & NoAxes()
VlnPlot(norm.co.seu, features = "PDGFRB", cols = mycoloursP)
```
9) Cluster 2 & Cluster 7 could be characterised as Immature Neurons
```{r}
FeaturePlot(neu, features = "TUBB3", label = TRUE) & NoAxes() #early neuronal marker
```
10) Cluster 0, Cluster 1, Cluster 3, Cluster 9, Cluster 6 are positive for several neuronal markers; at a later stage these clusters can be subsetted seperately to characterised the various neuronal types extensively
```{r fig.width=7, fig.height=15, fig.fullwidth=TRUE}
FeaturePlot(neu, features = c("STMN2", "MAP2", "DCX", "SOX11", "SYP", "NEFL", "STMN1", "ASCL1", "SLC32A1", "GAD1", "GAD2", "RBFOX1", "RBFOX3"), ncol = 2) & NoAxes()
```
```{r message=FALSE}
sessionInfo()
```