Changes between Version 22 and Version 23 of SOP/scRNA-seq

Timestamp:

08/25/20 17:38:26 (4 years ago)

Author:

ibarrasa

Comment:

--

SOP/scRNA-seq

@@ -102 +102 @@
 {{{
 all_Filt <- RunPCA(object = all_Filt, features = VariableFeatures(object = all_Filt))
+
 pdf("./PCAPlot.pdf", w=11, h=8.5)
 DimPlot(object = all_Filt, reduction = "pca")
@@ -116 +117 @@
 all_Filt <- RunUMAP(object = all_Filt, dims = 1:20)
 all_Filt <- RunTSNE(object = all_Filt, dims = 1:20)
+
 pdf("./UMAP_colorByExp.pdf", w=11, h=8.5)
 DimPlot(object = all_Filt, reduction = "umap")
@@ -129 +131 @@
 all_Filt <- FindNeighbors(object = all_Filt, dims = 1:20)
 all_Filt <- FindClusters(object = all_Filt, resolution = 0.5)
+
 pdf("./UMAP_colorByCluster_Res0.5.pdf", w=11, h=8.5)
 UMAPPlot(object = all_Filt, label= TRUE)
 dev.off()
+
 all_Filt <- FindClusters(object = all_Filt, resolution = 0.4)
 pdf("./UMAP_colorByCluster_Res0.4.pdf", w=11, h=8.5)
 UMAPPlot(object = all_Filt, label= TRUE)
 dev.off()
+
 all <- FindClusters(object = all_Filt, resolution = 0.3)
 pdf("./UMAP_colorByCluster_Res0.3.pdf", w=11, h=8.5)
@@ -148 +153 @@
   * consider each cell as a sample
   * aggregate counts across all cells in a group/cluster, and treat them as one sample
-
+Seurat has 2 functions "FindAllMarkers" and "FindMarkers" that work well as long as the fold change and percentage of cells expressing the gene thresholds are not relaxed from the defaults "logfc.threshold = 0.25, min.pct = 0.1." More stringent thresholds (MIN_LOGFOLD_CHANGE = 0.7,  MIN_PCT_CELLS_EXPR_GENE = .25) also work well.
+  * example command to compare each cluster to all other clusters:
+{{{
+all.markers.pos.wilcox = FindAllMarkers(all_Filt,
+                                        min.pct = MIN_PCT_CELLS_EXPR_GENE,
+                                        logfc.threshold = MIN_LOGFOLD_CHANGE,
+                                        only.pos = TRUE,
+                                        test.use="wilcox")
+}}}
+  * example command to compare 2 or more clusters or cell identities (''i.e.''  control and treatment) 
+{{{
+markers1_versus_18.pos.wilcox = FindMarkers(all_Filt,
+                                        min.pct = MIN_PCT_CELLS_EXPR_GENE,
+                                        logfc.threshold = MIN_LOGFOLD_CHANGE,
+                                        only.pos = TRUE,
+                                        test.use="wilcox",
+                                        ident.1  = "1",
+                                        ident.2 = "18"
+}}}
 === Add biological annotations to cells or cell clusters  ===