--- 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() ```