---
title: "Cluster Annotation"
author: "Nina-Lydia Kazakou"
date: "16/06/2021"
output: html_document
---
Here, I will use markers previously described in literature to roughly annotate the cell clusters present in this dataset, before subsetting and looking into the oligolineage.
# 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()
```
Neurons:
```{r fig.width=7, fig.height=5, fig.fullwidth=TRUE}
# General Markers
FeaturePlot(norm.co.seu, features = c("GPHN", "NCAM1", "NEFL", "NEUROD1"), order = TRUE, label = TRUE)
FeaturePlot(norm.co.seu, features = c( "RBFOX3", "RBFOX1", "MAP2", "SYP"), order = TRUE, label = TRUE)
FeaturePlot(norm.co.seu, features = c("SYNPR", "TUBB3"), order = TRUE, label = TRUE)
FeaturePlot(norm.co.seu, features = c("SNAP25", "STMN2", "RBFOX3", "GABRB2"), label = TRUE)
```
Inhibitory neurons:
```{r fig.width=7, fig.height=5, fig.fullwidth=TRUE}
FeaturePlot(norm.co.seu, features = c("GAD1", "GAD2", "SLC32A1", "PVALB"), label = TRUE)
FeaturePlot(norm.co.seu, features = c("ADARB2", "VIP", "LAMP5", "LHX6"), label = TRUE)
```
```{r fig.width=7, fig.height=23, fig.fullwidth=TRUE}
FeaturePlot(norm.co.seu, features = c("LAMA3", "VIP", "TAC3", "RYR3", "TSHZ2",
"RELN", "IL1RAPL2", "CXCL14", "EYA4",
"FBXL7", "KIT", "MEIS2", "PBX3", "PLCL1",
"MYO5B", "TRHDE", "PLCH1"), ncol = 2, label = TRUE)
```
Excitatory neurons
```{r fig.width=7, fig.height=5, fig.fullwidth=TRUE}
FeaturePlot(norm.co.seu, features = c("SATB2", "SLC12A6", "SLC17A7"), label = TRUE)
FeaturePlot(norm.co.seu, features = c("FEZF2", "RORB", "THEMIS"), label = TRUE)
```
Glutaminergic neurons:
```{r fig.width=7, fig.height=5, fig.fullwidth=TRUE}
FeaturePlot(norm.co.seu, features = c("GLS", "GRIN1", "GRIN2B", "SLC17A6"), label = TRUE)
FeaturePlot(norm.co.seu, features = c("VGLUT2", "SLC17A7", "VGLUT1"), label = TRUE)
```
Serotonergic neurons:
```{r fig.width=7, fig.height=5, fig.fullwidth=TRUE}
FeaturePlot(norm.co.seu, features = c("SLC6A4", "TPH1"), label = TRUE)
```
Neuronal receptors:
```{r fig.width=7, fig.height=5, fig.fullwidth=TRUE}
FeaturePlot(norm.co.seu, features = c("GABBR1", "SLC32A1", "VGAT"), label = TRUE)
```
Radial Glia & Neuroprogenitors:
```{r fig.width=7, fig.height=5, fig.fullwidth=TRUE}
# Radial Glia:
FeaturePlot(norm.co.seu, features = c("SLC1A3", "PAX6", "SOX2", "PDGFD", "GLI3"), label = TRUE, ncol = 3)
FeaturePlot(norm.co.seu, features = c("GLI3", "VIM", "HES1", "HES5"), label = TRUE)
#Outer Radial Glia:
FeaturePlot(norm.co.seu, features = c("TNC", "PTPRZ1", "FAM107A", "HOPX", "LIFR"), label = TRUE)
FeaturePlot(norm.co.seu, features = c("LIFR", "STAT3"), label = TRUE)
#Basal Radial Glia:
FeaturePlot(norm.co.seu, features = c("VIM", "SOX2", "PAX6", "HOPX"), label = TRUE)
#Ventricular Radial Glia:
FeaturePlot(norm.co.seu, features = c("CRYAB", "PDGFD", "TAGLN2", "FBXO32", "PALLD"), label = TRUE)
#Early neuronal markers:
FeaturePlot(norm.co.seu, features = c("EOMES", "ELAVL4", "NEUROG1", "NEUROD1"), label = TRUE)
FeaturePlot(norm.co.seu, features = c("NEUROD4", "PPP1R17", "PENK"), label = TRUE)
#From Radial Glia to Intermediate Progenitors to Neurons
FeaturePlot(norm.co.seu, features = c("SLC1A3", "PAX6", "VIM", "GFAP"), label = TRUE)
FeaturePlot(norm.co.seu, features = c("RBFOX1"), label = TRUE)
```
Astrocytes:
```{r fig.width=7, fig.height=25, fig.fullwidth=TRUE}
FeaturePlot(norm.co.seu, features = c("S100B",
"SLC1A3",
"EFNB3",
"TFF3",
"SPARCL1",
"SPON1",
"TGFB2",
"GJA1",
"AQP4",
"GLUL",
"SOX9",
"NDRG2",
"GFAP",
"ALDH1A1",
"APOE",
"FGFR3"), label = TRUE, ncol = 2)
```
```{r fig.width=7, fig.height=35, fig.fullwidth=TRUE}
FeaturePlot(norm.co.seu, features = c("HPSE2",
"TENM2",
"ZNF98",
"CCDC85A",
"SLC38A1",
"GALNT15",
"CFAP299",
"SPAG17",
"DTHD1",
"ADGB",
"UAP1",
"SPOCD1",
"CLCF1",
"CDH19",
"SORCS1",
"SAMHD1",
"NRGN",
"CAMK2A",
"THY1",
"ENC1",
"SYT1",
"CALM3",
"ST18",
"CTNNA3",
"APLNR"), label = TRUE, ncol = 2)
```
Pericytes:
```{r fig.width=7, fig.height=5, fig.fullwidth=TRUE}
FeaturePlot(norm.co.seu, features = c("PDGFRB", "COL1A1", "COL1A2", "COL1A3"), label = TRUE)
FeaturePlot(norm.co.seu, features = c("ACTA2", "SPARC", "BGN", "S100A10"), label = TRUE)
```
Microglia:
```{r fig.width=7, fig.height=5, fig.fullwidth=TRUE}
FeaturePlot(norm.co.seu, features = c("TMEM119", "CD11B", "CD45", "IBA1", "CX3CR1", "F4/80", "CD68", "CD40"), label = TRUE)
```
Oligodendroglia:
```{r fig.width=7, fig.height=5, fig.fullwidth=TRUE}
#General Glia Markers
FeaturePlot(norm.co.seu, features = c("OLIG1", "OLIG2", "SOX10"), label = TRUE)
FeaturePlot(norm.co.seu, features = c("CD9", "PPP1R16B", "SOX8"), label = TRUE)
#Oligo lineage progression
FeaturePlot(norm.co.seu, features = c("MYRF", "O4", "GPR56", "GPR17"), label = TRUE)
#OPCs
FeaturePlot(norm.co.seu, features = c("PDGFRA", "PCDH15", "BCAN", "SOX6"), label = TRUE)
FeaturePlot(norm.co.seu, features = c("FOXJ3", "ZNF238"), label = TRUE) #maintenance of mitotic OPCs
FeaturePlot(norm.co.seu, features = c("NELL1", "PAX3"), label = TRUE) #Spinal Cord OPCs from HCA dataset
#COPs
FeaturePlot(norm.co.seu, features = c("ETV1", "CHST9", "MYT1", "TENM2"), label = TRUE)
FeaturePlot(norm.co.seu, features = c("CAMK2A", "KCNQ5", "SEMA5B", "SYT1"), label = TRUE)
FeaturePlot(norm.co.seu, features = c("GPR17", "BMPER", "EPHB1", "ARHGAP24"), label = TRUE)
FeaturePlot(norm.co.seu, features = c("DOCK8", "APBB1IP"), label = TRUE)
#Oligodendrocytes
FeaturePlot(norm.co.seu, features = c("MBP", "PLP1", "CNP", "MAG"), label = TRUE)
FeaturePlot(norm.co.seu, features = c("MOG", "OSP", "BCAS1", "ZNF488"), label = TRUE)
FeaturePlot(norm.co.seu, features = c("GALC", "MOBP", "ZNF536"), label = TRUE)
```
Summarized markers used to characterise the monolayer dataset:
```{r fig.width=7, fig.height=5, fig.fullwidth=TRUE}
# Radial Glia
FeaturePlot(norm.co.seu, features = c("VIM", "PAX6", "HES1"), label = TRUE)
# Outer Radial Glia
FeaturePlot(norm.co.seu, features = c("HOPX", "PTPRZ1"), label = TRUE)
# Pre-OPCs
FeaturePlot(norm.co.seu, features = c("SPARCL1", "BCAN"), label = TRUE)
# Astocytes (mature)
FeaturePlot(norm.co.seu, features = c("AQP4", "TAGLN2", "SPON1"), label = TRUE)
# Oligodendroglia
FeaturePlot(norm.co.seu, features = c("OLIG1", "OLIG2", "SOX10"), label = TRUE)
# Neurons
FeaturePlot(norm.co.seu, features = c("DCX", "SOX11", "STMN2"), label = TRUE)
# Pericytes
FeaturePlot(norm.co.seu, features = c("ACTA2", "AXNA2"), label = TRUE)
```
Markers previously identified in our datasets:
```{r fig.width=7, fig.height=5, fig.fullwidth=TRUE}
FeaturePlot(norm.co.seu, features = c("RBFOX1", "SPARC", "OPALIN"), label = TRUE)
FeaturePlot(norm.co.seu, features = c("PAX3", "NELL1", "KLK6"), label = TRUE)
```
```{r}
sessionInfo()
```