Data Visualization (Ragas)-L2

1. load libraries

2. Load Seurat Object

L2 <- readRDS("../0-RDS_Cell_lines/L2_clustered.rds")

L2 <- NormalizeData(L2, normalization.method = "LogNormalize", scale.factor = 10000)

Avis : The `slot` argument of `SetAssayData()` is deprecated as of SeuratObject 5.0.0.
Please use the `layer` argument instead.Avis : The `slot` argument of `GetAssayData()` is deprecated as of SeuratObject 5.0.0.
Please use the `layer` argument instead.Performing log-normalization
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UMAP colored by cell type and expression - dittoDimPlot

spe <- L2

 DefaultAssay(spe) = "RNA"

library(Ragas)


RunDimPlot(object = spe, group.by = "SCT_snn_res.0.2")

RunDimPlot(object = spe, group.by = "SCT_snn_res.0.3")

DimPlot(spe, reduction = "umap", group.by = "cell_line",label = T, label.box = T)

DimPlot(spe, reduction = "umap", group.by = "SCT_snn_res.0.2",label = T, label.box = T)

DimPlot(spe, reduction = "umap", group.by = "predicted.celltype.l2",label = T, label.box = T, repel = T)

FeaturePlot(L2, features = "CCR7", reduction = "umap") ## TCM

Avis : The `slot` argument of `FetchData()` is deprecated as of SeuratObject 5.0.0.
Please use the `layer` argument instead.

FeaturePlot(L2, features = "GZMK", reduction = "umap") ## Th1

FeaturePlot(L2, features = "IL17RB", reduction = "umap") ## Th2

Avis : All cells have the same value (0) of “IL17RB”

FeaturePlot(L2, features = "CTSH", reduction = "umap") ## Th17

Avis : All cells have the same value (0) of “CTSH”

FeaturePlot(L2, features = "CCR10", reduction = "umap") ## Th22

FeaturePlot(L2, features = c("IL2RA", "FOXP3"), reduction = "umap") ## Th22

NA
NA

Create a Pi object

#rm(All_samples_Merged)

my.pbmc.pi <- CreatePostIntegrationObject(object = spe)

Post-integration object created

RunDimPlot(object = my.pbmc.pi, group.by = "SCT_snn_res.0.2")

my.pbmc.pi

An object of class Pi 
6 fields in the object: seurat.obj, exp.freq, markers, ds, cell.prop, parent.meta.data.
The following field has been processed:
    seurat.obj: A Seurat object of 36601 features and 5935 cells.
        6 assays: RNA, ADT, prediction.score.celltype.l1, prediction.score.celltype.l2, prediction.score.celltype.l3, SCT, and 5 reductions: integrated_dr, ref.umap, pca, umap, harmony
Metadata from the parent object provided? No 
Subclusters integrated? No

2. Marker gene identification

#rm(spe)

my.pbmc.pi <- RunFindAllMarkers(my.pbmc.pi, 
                               logfc.threshold = 0.1,
                               min.pct = 0.1,
                               min.diff.pct = 0.2,
                               only.pos = TRUE,
                                assay = "RNA",
                               ident = "SCT_snn_res.0.2")

Calculating cluster 0
Calculating cluster 1
Calculating cluster 2
PiData Markers|SCT_snn_res.0.2|AllMarkers|test.use=wilcox already exisits. Overwriting...

2. Marker gene Visualization

p1 <- RunMatrixPlot(my.pbmc.pi,
              markers.key = "Markers|SCT_snn_res.0.2|AllMarkers|test.use=wilcox", 
              column.anno.name.rot = 45, 
              heatmap.height = 8)

Set active identity to SCT_snn_res.0.2
Performing relative-counts-normalization
Centering and scaling data matrix

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p1