Added analysis scripts and notebooks.

SampleSheet.csv

+Lane,Sample,Index,Group
+*,FB-10-A4,SI-GA-G1,AB_Ctrl
+*,FB-11-C0,SI-GA-G2,CD_High
+*,FB-12-F1,SI-GA-G3,EF_Low
+*,FB-16-B1,SI-GA-G4,AB_Ctrl
+*,FB-17-E3,SI-GA-G5,EF_Low
+*,FB-18-F2,SI-GA-G6,EF_Low
+*,FB-4-B3,SI-GA-G7,AB_Ctrl
+*,FB-5-C1,SI-GA-G8,CD_High
+*,FB-6-D1,SI-GA-G9,CD_High

scripts/5_prepareSCVelo.R

+library(SeuratWrappers)
+library(Seurat)
+library(SeuratDisk)
+
+ts <- c("FB_10-A4_T", "FB_12-F1_T", "FB_16-B1_T", "FB_18-F2_T", "FB_4-B3_T", "FB_6-D1_T", "FB_11-C0_T", "FB_17-E3_T", "FB_5-C1_T")
+veldata <- list()
+velobj <- list()
+for (i in ts) {
+    veldata[[i]] <- ReadVelocity(file = paste0("Birocchi_SciTraMed2022/velocyto_loom/", i, ".loom"))
+    velobj[[i]] <- as.Seurat(x = veldata[[i]])
+    velobj[[i]] <- RenameCells(object = velobj[[i]], new.names = gsub(pattern = "merged", replacement = "", x = colnames(velobj[[i]])))
+    velobj[[i]] <- RenameCells(object = velobj[[i]], new.names = gsub(pattern = "x$", perl = T, replacement = "", x = colnames(velobj[[i]])))
+    velobj[[i]] <- RenameCells(object = velobj[[i]], new.names = gsub(pattern = ":", perl = T, replacement = "_", x = colnames(velobj[[i]])))
+    velobj[[i]] <- RenameCells(object = velobj[[i]], new.names = gsub(pattern = "_", perl = T, replacement = "-", x = colnames(velobj[[i]])))
+    velobj[[i]] <- RenameCells(object = velobj[[i]], new.names = gsub(pattern = "-T-", perl = T, replacement = "_", x = colnames(velobj[[i]])))
+    velobj[[i]]@meta.data$orig.ident <- i
+}
+
+velobj_ctrl <- merge(merge(velobj[["FB_10-A4_T"]], velobj[["FB_16-B1_T"]]), velobj[["FB_4-B3_T"]])
+velobj_high <- merge(merge(velobj[["FB_11-C0_T"]], velobj[["FB_5-C1_T"]]), velobj[["FB_6-D1_T"]])
+velobj_low <- merge(merge(velobj[["FB_12-F1_T"]], velobj[["FB_18-F2_T"]]), velobj[["FB_17-E3_T"]])
+velo_full <- merge(merge(velobj_ctrl, velobj_high), velobj_low)
+
+wdir <- "Birocchi_SciTraMed2022/results"
+macro_obj <- readRDS(paste(wdir, "MacrophagesFiltered", "01-fastMNN_2", "MacrophagesFiltered_fastMNN_final.rds", sep = "/"))
+colnames(macro_obj)[1:5]
+
+# write.csv(Cells(macro_obj), file = paste(wdir, "MacrophagesFiltered", "01-fastMNN_2", "MacrophagesFiltered_fastMNN_cellID_obs.csv", sep = "/"), row.names = FALSE)
+# write.csv(Embeddings(macro_obj, reduction = "umap"), file = paste(wdir, "MacrophagesFiltered", "01-fastMNN_2", "MacrophagesFiltered_fastMNN_cell_embeddings.csv", sep = "/"))
+# colnames(macro_obj@meta.data)
+# write.csv(macro_obj@meta.data[,c("orig.ident", "RNA_Group", "RNA_snn_res.0.6", "RNA_snn_res.1", "RNA_snn_res.1.2")],
+#           file = paste(wdir, "MacrophagesFiltered", "01-fastMNN_2", "MacrophagesFiltered_fastMNN_mdata.csv", sep = "/"))
+# 
+# #write.csv(Cells(obj), file = "cellID_obs.csv")
+# macro_obj_embedding <- Embeddings(macro_obj, reduction = "umap")
+# head(macro_obj_embedding)
+# 
+# #write.csv(Embeddings(obj, reduction = "umap"), file = "cell_embeddings.csv")
+# #my_clusters <- obj@meta.data$RNA_snn_res.0.6
+# macro_obj_clu <- FetchData(object = macro_obj, vars = "RNA_snn_res.0.6")
+# colnames(macro_obj_clu) <- c("Seurat_RNA_snn_res.0.6")
+# head(macro_obj_clu)
+# #write.csv(obj@meta.data$seurat_clusters, file = "clusters.csv")
+# macro_obj_groups <- FetchData(object = macro_obj, "RNA_Group")
+# colnames(macro_obj_groups) <- c("RNA_Group")
+# head(macro_obj_groups)
+# 
+# cells <- intersect(Cells(velo_full), Cells(macro_obj))
+# length(Cells(velo_full))
+# length(Cells(macro_obj))
+# length(cells)
+# velo_full <- subset(velo_full, cells = cells)
+# 
+# velo_full[["RNA"]] <- velo_full[["spliced"]]
+# #########velo_full[["RNA"]] <- macro_obj@assays$RNA
+# # adding clusters and groups
+# velo_full <- AddMetaData(object = velo_full, metadata = macro_obj_clu)
+# velo_full <- AddMetaData(object = velo_full, metadata = macro_obj_groups)
+# 
+# colnames(velo_full@meta.data)
+# 
+# names(velo_full@assays)
+# str(velo_full)
+# # creating fake umap slots in order to create the reduction slot associated
+# velo_full <- SCTransform(velo_full)
+# velo_full <- RunPCA(velo_full)
+# velo_full <- RunUMAP(velo_full, dims = 1:30, n.components = 2)
+# velo_full <- FindNeighbors(velo_full, dims = 1:30)
+# velo_full <- FindClusters(velo_full)
+# DefaultAssay(velo_full) <- "RNA"
+# # restore original UMAPs
+# head(velo_full@reductions$umap@cell.embeddings)
+# head(macro_obj_embedding[rownames(velo_full@reductions$umap@cell.embeddings),])
+# velo_full@reductions$umap@cell.embeddings <- macro_obj_embedding[rownames(velo_full@reductions$umap@cell.embeddings),]
+# SaveH5Seurat(velo_full, filename = paste(wdir, "MacrophagesFiltered", "01-fastMNN_2","MacrophagesFiltered_velo.h5Seurat", sep = "/"), overwrite = T)
+# Convert(paste(wdir, "MacrophagesFiltered", "01-fastMNN_2","MacrophagesFiltered_velo.h5Seurat", sep = "/"), dest = "h5ad", overwrite = T)
+
+# Groups
+Idents(macro_obj) <- "RNA_Group"
+velobj_groups <- list("AB_Ctrl" = velobj_ctrl, "CD_High" = velobj_high, "EF_Low" = velobj_low)
+for (group in names(velobj_groups)) {
+    print(group)
+    velo_sub <- velobj_groups[[group]]
+    macro_obj_sub <- subset(macro_obj, idents = group)
+    macro_obj_sub_embedding <- Embeddings(macro_obj_sub, reduction = "umap")
+    head(macro_obj_sub_embedding)
+    macro_obj_sub_clu <- FetchData(object = macro_obj_sub, vars = "RNA_snn_res.0.6")
+    head(macro_obj_sub_clu)
+    cells_sub <- intersect(Cells(velo_sub), Cells(macro_obj_sub))
+    length(Cells(velo_sub))
+    length(Cells(macro_obj_sub))
+    length(cells_sub)
+    velo_sub <- subset(velo_sub, cells = cells_sub)
+    velo_sub[["RNA"]] <- velo_sub[["spliced"]]
+    # adding clusters
+    velo_sub <- AddMetaData(object = velo_sub, metadata = macro_obj_sub_clu)
+    # creating fake umap slots in order to create the reduction slot associated
+    velo_sub <- SCTransform(velo_sub)
+    velo_sub <- RunPCA(velo_sub)
+    velo_sub <- RunUMAP(velo_sub, dims = 1:30)
+    velo_sub <- FindNeighbors(velo_sub, dims = 1:30)
+    velo_sub <- FindClusters(velo_sub)
+    DefaultAssay(velo_sub) <- "RNA"
+    # restore original UMAPs
+    head(velo_sub@reductions$umap@cell.embeddings)
+    head(macro_obj_sub_embedding[rownames(velo_sub@reductions$umap@cell.embeddings),])
+    velo_sub@reductions$umap@cell.embeddings <- macro_obj_sub_embedding[rownames(velo_sub@reductions$umap@cell.embeddings),]
+    SaveH5Seurat(velo_sub, filename = paste(wdir, "MacrophagesFiltered", "01-fastMNN_2", paste0("MacrophagesFiltered-", group, "_velo.h5Seurat"), sep = "/"), overwrite = T)
+    Convert(paste(wdir, "MacrophagesFiltered", "01-fastMNN_2", paste0("MacrophagesFiltered-", group, "_velo.h5Seurat"), sep = "/"), dest = "h5ad", overwrite = T)
+}