Hello I'm graduate student who try to analyze single cell. I analyze this data for finding aging markers. But there's no one to help me....

So I don't know whats wrong in my code Please help me. So my final goal is find aging markers but results showed there's no aging markers in my data.

But the Dimplot(res=0.6) shows me there's definite difference between Young and Old type...

I don't know how can handle this problem

I'm so desperate.

Thank you!

young1 <-readRDS("~/Desktop/GSE137869/GSE137869_aging/WAT_M_Y.rds")
young2 <-readRDS("~/Desktop/GSE137869/GSE137869_aging//WAT_F_Y/WAT_F_Y.rds")
old1 <- readRDS("~/Desktop/GSE137869/GSE137869_aging/WAT_M_O/WAT_M_O.rds")
old2 <- readRDS("~/Desktop/GSE137869/GSE137869_aging//WAT_F_O/WAT_F_O.rds")

BiocManager::install('glmGamPoi')
library(glmGamPoi)

WAT <- merge(young1, y = c(young2, old1, old2), 
             add.cell.ids = c("young1", "young2", "old1", "old2"),
             project = "WAT") %>%
  NormalizeData() %>%
  FindVariableFeatures(selection.method = "vst", nfeatures = 2000)%>%
  ScaleData()%>%
  SCTransform(vars.to.regress = c("percent.mt"))

WAT <- RunPCA(WAT, assay = "SCT", npcs = 50)


WAT@meta.data$type <- c(rep("Young", ncol(young1)),
                        rep("Young", ncol(young2)),
                        rep("Old", ncol(old1)),
                        rep("Old", ncol(old2)))

View(WAT@meta.data)


harmonized_WAT <- RunHarmony(WAT,
                             group.by = "type",
                             reduction ="pca",
                             assay.use = "SCT",
                             reduction.save = "harmony")

harmonized_WAT <- RunUMAP(harmonized_WAT, reduction = "harmony",
                          assay = "SCT", dims = 1:30)

harmonized_WAT <- FindNeighbors(object = harmonized_WAT, reduction = "harmony")
#harmonized_WAT <- FindClusters(harmonized_WAT, resolution = c(0.6))
harmonized_WAT <- FindClusters(harmonized_WAT, resolution = c(0.2, 0.4, 0.6, 0.8, 1.0, 1.2))

View(harmonized_WAT@meta.data)


Idents(object = harmonized_WAT) <- "SCT_snn_res.0.6"
DimPlot(harmonized_WAT, 
        label = TRUE, 
        split.by = "type")


harmonized_WAT <- PrepSCTFindMarkers(harmonized_WAT)

devtools::install_github('immunogenomics/presto')

View(harmonized_WAT@meta.data)

WAT_aging_markers <- FindAllMarkers(object = harmonized_WAT,
                          only.pos = TRUE,
                          assay = "SCT",
                          group.by = "type",
                          logfc.threshold = 0.5,
                          min.pct = 0.05,
                          test.use = "wilcox")

This is my dataset

This is Dimplot I draw