astrocyte_integration_subclustering_and_DE.Rmd

---
title: "Astrocyte Integration, subclustering, Differential expression"
date: "2023-07-25"
output: html_document
---

```{r setup, include=FALSE}
knitr::opts_chunk$set(echo = TRUE)
```

The following script was run on a GPU cluster. This is an example script for EC Astrocytes integration and processing. The same script was used for processing other brain regions. Processed data required as input for the script will be available upon request. 
```{r}
integrate_f = file.path(rdata_dir, "EC_astrocyte_integrated.Rdata")
# load data
EC_f = file.path(rdata_dir, "EC_rPCA.Rdata")
load(EC_f)
EC = EC_rPCA
rm(EC_rPCA)

# Subset astrocytes
EC$celltype = EC$Annie_Cell_Type
Idents(EC) = "celltype"
ast = EC[, EC$celltype == "astrocyte"]
DimPlot(ast)

# CCA integration
DefaultAssay(ast) = "RNA"
ast_list = SplitObject(ast, split.by = "Donor.ID")
n_HVG = 2000
ast_list = lapply(X = ast_list, FUN = function(x) {
    x = NormalizeData(x)
    x = FindVariableFeatures(x, selection.method = "vst", nfeatures = n_HVG)
})
features = SelectIntegrationFeatures(object.list = ast_list, nfeatures = n_HVG)
anchors = FindIntegrationAnchors(object.list = ast_list, anchor.features = features)
save(anchors, file = integrate_f)
EC_ast_cca = IntegrateData(anchorset = anchors)
save(EC_ast_cca, file = integrate_f)

# subclustering
DefaultAssay(EC_ast_cca) = "integrated"
EC_ast_cca = ScaleData(EC_ast_cca, verbose = FALSE)
EC_ast_cca = RunPCA(EC_ast_cca, npcs = 50, verbose = FALSE)
ElbowPlot(EC_ast_cca, ndims = 50)
EC_ast_cca = RunUMAP(EC_ast_cca, reduction = "pca", dims = 1:npc)
EC_ast_cca = FindNeighbors(EC_ast_cca, reduction = "pca", dims = 1:npc)
EC_ast_cca = FindClusters(EC_ast_cca, resolution = seq(0.1, 1, 0.1))
save(EC_ast_cca, file = integrate_f)

# Markers
marker_file = "../results/marker.csv"
markers = FindAllMarkers(ast_CCA, min.pct = 0, logfc.threshold = 0, min.cells.feature = 0,
                           test.use = "LR", return.thresh = 1, latent.vars = "Donor.ID")
markers = df_to_dt(markers)
fwrite(markers, marker_file)
```