FDR analysis - Results - thresholds true 3% 10%

Author

Silvia Giulia Galfrè

Published

June 16, 2024

Preamble

library(COTAN)
library(pROC)
options(parallelly.fork.enable = TRUE)
library(Seurat)
library(monocle3)
library(reticulate)
library(cowplot)
library(stringr)
library(dplyr)
library(tidyr)
library(ggplot2)
library(ggpubr)

methods.color <- c("COTAN"="#F73604","Seurat"="#ABD9E9","Seurat_scTr"="#74ADD1","Seurat_Bimod"="#4575B4", "Monocle"="#DAABE9", "Scanpy"="#7F9B5C" )

dirOut <- "Results/FDR/"
if (!dir.exists(dirOut)) {
  dir.create(dirOut)
}

dataSetDir <- "Data/MouseCortexFromLoom/FDR/MergedClusters_For_FDR/"

Dataset composition

datasets_csv <- read.csv(file.path(dataSetDir,"Cells_Usage_DataFrame.csv"),
                         row.names = 1
                        ) 

datasets_csv[1:3,]
                 Group            Collection E13.5.432 E13.5.187 E13.5.434
1 2_Clusters_even_near E13.5-434_+_E15.0-428         0         0       318
2 2_Clusters_even_near E15.0-432_+_E13.5-432       536         0         0
3 2_Clusters_even_near E15.0-508_+_E15.0-509         0         0         0
  E13.5.184 E13.5.437 E13.5.510 E15.0.432 E15.0.509 E15.0.510 E15.0.508
1         0         0         0         0         0         0         0
2         0         0         0       536         0         0         0
3         0         0         0         0       397         0       397
  E15.0.428 E15.0.434 E15.0.437 E17.5.516 E17.5.505
1       318         0         0         0         0
2         0         0         0         0         0
3         0         0         0         0         0

Define which genes are expressed

For each data set we need to define, independently from the DEA methods, which genes are specific for each cluster. So we need to define first which genes are expressed and which are not expressed. To do so we can take advantage from the fact that we have the original clusters from which the cells were sampled to create the artificial datasets. So looking to the original cluster we define as expressed all genes present in at least the 10% of cells and we define as not expressed the genes completely absent or expressed in less than 3% of cells.

Since these two thresholds can have a big influence on the tools performances we will test also others in other pages.

file.presence <- readRDS("Data/MouseCortexFromLoom/FDR/Results/GenePresence_PerCluster.RDS")

for (file in list.files("Data/MouseCortexFromLoom/SingleClusterRawData/")) {
#  print(file)
  Code <- str_split(file,pattern = "_",simplify = T)[1]
  Time <- str_split(Code,pattern = "e",simplify = T)[2]
  Cluster <- str_split(Code,pattern = "e",simplify = T)[1]
  Cluster <- str_remove(Cluster,pattern = "Cl")
  Cluster <- paste0("E",Time,"-",Cluster)
  file.presence[,Cluster] <- "Absent"
  dataset.cl <- readRDS(file.path("Data/MouseCortexFromLoom/SingleClusterRawData/",
                                         file))
  number.cell.expressing <- rowSums(dataset.cl > 0)
  AbsentThreshold <- round(0.03*dim(dataset.cl)[2],digits = 0)
  PresenceThreshold <- round(0.1*dim(dataset.cl)[2],digits = 0)
  file.presence[names(number.cell.expressing[number.cell.expressing > AbsentThreshold]),Cluster] <- "Uncertain" 
  
  file.presence[names(number.cell.expressing[number.cell.expressing >= PresenceThreshold]),Cluster] <- "Present"
  print(Cluster)
  print(table(file.presence[,Cluster]))
  
  }
[1] "E13.5-184"

   Absent   Present Uncertain 
     5519      6027      3149 
[1] "E13.5-187"

   Absent   Present Uncertain 
     4926      7151      2618 
[1] "E15.0-428"

   Absent   Present Uncertain 
     6386      5253      3056 
[1] "E13.5-432"

   Absent   Present Uncertain 
     5765      5975      2955 
[1] "E15.0-432"

   Absent   Present Uncertain 
     5912      5843      2940 
[1] "E13.5-434"

   Absent   Present Uncertain 
     6124      5487      3084 
[1] "E15.0-434"

   Absent   Present Uncertain 
     6301      5396      2998 
[1] "E13.5-437"

   Absent   Present Uncertain 
     5943      5783      2969 
[1] "E15.0-437"

   Absent   Present Uncertain 
     6017      5713      2965 
[1] "E17.5-505"

   Absent   Present Uncertain 
     6048      5662      2985 
[1] "E15.0-508"

   Absent   Present Uncertain 
     5625      6240      2830 
[1] "E15.0-509"

   Absent   Present Uncertain 
     5452      6454      2789 
[1] "E13.5-510"

   Absent   Present Uncertain 
     4924      6958      2813 
[1] "E15.0-510"

   Absent   Present Uncertain 
     5070      6952      2673 
[1] "E17.5-516"

   Absent   Present Uncertain 
     5821      5847      3027 

2 Clusters even

2_Clusters_even_near

True vector

subset.datasets_csv <-datasets_csv[datasets_csv$Group == "2_Clusters_even_near",] 

ground_truth_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  clusters <- str_split(subset.datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  file.presence.subset <- file.presence[,clusters]
  
  
  #file.presence.subset <- as.matrix(file.presence.subset)
  
  
  ground_truth <- as.data.frame(matrix(nrow = nrow(file.presence.subset),
                                       ncol = ncol(file.presence.subset)))
  rownames(ground_truth) <- rownames(file.presence.subset)
  colnames(ground_truth) <- colnames(file.presence.subset)
  
  ground_truth[file.presence.subset == "Absent"] <- 0
  ground_truth[file.presence.subset == "Present"] <- 1
  ground_truth[file.presence.subset == "Uncertain"] <- 0.6
  
  file.presence.subset <- ground_truth
  
  for (col in 1:ncol(ground_truth)) {
    ground_truth[,col] <- FALSE  
    ground_truth[file.presence.subset[,col] == 1 & rowMeans(file.presence.subset[,-col,drop = FALSE]) < 0.35  ,col] <- TRUE
    }
  
  ground_truth$genes <- rownames(ground_truth) 
  ground_truth <- pivot_longer(ground_truth,
                               cols = 1:(ncol(ground_truth)-1),
                               names_to = "clusters")
  ground_truth$data_set <- subset.datasets_csv[ind,1]
  ground_truth$set_number <- ind 
  ground_truth_tot <- rbind(ground_truth_tot, ground_truth)
  
}
ground_truth_tot <- ground_truth_tot[2:nrow(ground_truth_tot),]

head(ground_truth_tot,20)
# A tibble: 20 × 5
   genes  clusters  value data_set             set_number
   <chr>  <chr>     <lgl> <chr>                     <int>
 1 Neil2  E13.5-434 FALSE 2_Clusters_even_near          1
 2 Neil2  E15.0-428 FALSE 2_Clusters_even_near          1
 3 Lamc1  E13.5-434 FALSE 2_Clusters_even_near          1
 4 Lamc1  E15.0-428 FALSE 2_Clusters_even_near          1
 5 Lama1  E13.5-434 FALSE 2_Clusters_even_near          1
 6 Lama1  E15.0-428 FALSE 2_Clusters_even_near          1
 7 Hs3st1 E13.5-434 FALSE 2_Clusters_even_near          1
 8 Hs3st1 E15.0-428 FALSE 2_Clusters_even_near          1
 9 Fabp3  E13.5-434 FALSE 2_Clusters_even_near          1
10 Fabp3  E15.0-428 FALSE 2_Clusters_even_near          1
11 Nrg2   E13.5-434 FALSE 2_Clusters_even_near          1
12 Nrg2   E15.0-428 FALSE 2_Clusters_even_near          1
13 Kdelr3 E13.5-434 FALSE 2_Clusters_even_near          1
14 Kdelr3 E15.0-428 FALSE 2_Clusters_even_near          1
15 Bend4  E13.5-434 FALSE 2_Clusters_even_near          1
16 Bend4  E15.0-428 FALSE 2_Clusters_even_near          1
17 Gjb4   E13.5-434 FALSE 2_Clusters_even_near          1
18 Gjb4   E15.0-428 FALSE 2_Clusters_even_near          1
19 Mogs   E13.5-434 FALSE 2_Clusters_even_near          1
20 Mogs   E15.0-428 FALSE 2_Clusters_even_near          1
length(unique(ground_truth_tot$genes))
[1] 14695
sum(ground_truth_tot$value)
[1] 24

ROC for COTAN

onlyPositive.pVal.Cotan_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",
                      subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))
  deaCOTAN <- getClusterizationData(dataset,clName = "mergedClusters")[[2]]
  pvalCOTAN <- pValueFromDEA(deaCOTAN,
              numCells = getNumCells(dataset),method = "none")
  
  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.names <- c(cl.names,
                  str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1])
    cl.names <- cl.names[!is.na(cl.names)]
  }
  colnames(deaCOTAN) <- cl.names
  colnames(pvalCOTAN) <- cl.names
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  onlyPositive.pVal.Cotan <- pvalCOTAN

    for (cl in cl.names) {
    print(cl)
    #temp.DEA.CotanSign <- deaCOTAN[rownames(pvalCOTAN[pvalCOTAN[,cl] < 0.05,]) ,]
    onlyPositive.pVal.Cotan[rownames(deaCOTAN[deaCOTAN[,cl] < 0,]),cl] <-  1 #onlyPositive.pVal.Cotan[rownames(deaCOTAN[deaCOTAN[,cl] < 0,]),cl]+1
    }
    
    onlyPositive.pVal.Cotan$genes <- rownames(onlyPositive.pVal.Cotan) 
    onlyPositive.pVal.Cotan <- pivot_longer(onlyPositive.pVal.Cotan,
                                            values_to = "p_values",
                               cols = 1:(ncol(onlyPositive.pVal.Cotan)-1),
                               names_to = "clusters")
  onlyPositive.pVal.Cotan$data_set <- subset.datasets_csv[ind,1]
  onlyPositive.pVal.Cotan$set_number <- ind 
  onlyPositive.pVal.Cotan_tot <- rbind(onlyPositive.pVal.Cotan_tot, onlyPositive.pVal.Cotan)
  
  }
[1] "E13.5-434"
[1] "E15.0-428"
[1] "E15.0-432"
[1] "E13.5-432"
[1] "E15.0-509"
[1] "E15.0-508"
onlyPositive.pVal.Cotan_tot <- onlyPositive.pVal.Cotan_tot[2:nrow(onlyPositive.pVal.Cotan_tot),]

onlyPositive.pVal.Cotan_tot <- merge.data.frame(onlyPositive.pVal.Cotan_tot,
                                                ground_truth_tot,by = c("genes","clusters","data_set","set_number"),all.x = T,all.y = F)

#onlyPositive.pVal.Cotan_tot <- onlyPositive.pVal.Cotan_tot[order(onlyPositive.pVal.Cotan_tot$p_values,
 #                                                                decreasing = F),]
# df <- as.data.frame(matrix(nrow = nrow(onlyPositive.pVal.Cotan_tot),ncol = 3)) 
# colnames(df) <- c("TPR","FPR","Method")
# df$Method <- "COTAN"
# 
# Positive <- sum(onlyPositive.pVal.Cotan_tot$value) 
# Negative <- sum(!onlyPositive.pVal.Cotan_tot$value)
# 
# for (i in 1:nrow(onlyPositive.pVal.Cotan_tot)) {
#   df[i,"TPR"] <- sum(onlyPositive.pVal.Cotan_tot[1:i,"value"])/Positive
#   df[i,"FPR"] <- (i-sum(onlyPositive.pVal.Cotan_tot[1:i,"value"]))/Negative
  
# Convert TRUE/FALSE to 1/0
onlyPositive.pVal.Cotan_tot$value <- as.numeric(onlyPositive.pVal.Cotan_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultCOTAN <- roc(onlyPositive.pVal.Cotan_tot$value, 1 - onlyPositive.pVal.Cotan_tot$p_values)

# Plot the ROC curve
#plot(roc_resultCOTAN)

ROC for Seurat

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

# Plot the ROC curve
#plot(roc_resultSeurat)

ROC for Seurat scTransform

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_ScTransform_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat_scTr <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

ROC for Seurat Bimod

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_Bimod_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat_Bimod <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

ROC for Monocle

deaMonocle_tot <- NA
for (ind in 1:dim(subset.datasets_csv)[1]) {
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))
  
  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  #print(file.code)
  
  deaMonocle <- read.csv(file.path(dirOut,paste0(file.code,"Monocle_DEA_genes.csv")),row.names = 1) 
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaMonocle[deaMonocle$cell_group == cl.val,"cell_group"] <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaMonocle) <- str_replace(colnames(deaMonocle),
                                     pattern = "cell_group",
                                     replacement = "clusters") 
  colnames(deaMonocle) <- str_replace(colnames(deaMonocle),
                                     pattern = "gene_id",
                                     replacement = "genes")
  
  deaMonocle$data_set <- subset.datasets_csv[ind,1]
  deaMonocle$set_number <- ind 
  deaMonocle <- as.data.frame(deaMonocle)
  deaMonocle_tot <- rbind(deaMonocle_tot, deaMonocle)
  
  
  }
 deaMonocle_tot <-  deaMonocle_tot[2:nrow(deaMonocle_tot),]
 
 deaMonocle_tot <- merge.data.frame(deaMonocle_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaMonocle_tot$value <- as.numeric(deaMonocle_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultMonocle <- roc(deaMonocle_tot$value, 1 - deaMonocle_tot$marker_test_p_value)

# Plot the ROC curve
#plot(roc_resultMonocle)

ROC from Scanpy

deaScanpy_tot <- NA
for (ind in 1:dim(subset.datasets_csv)[1]) {
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  #print(file.code)
  
  deaScanpy <- read.csv(file.path(dirOut,paste0(file.code,"Scanpy_DEA_genes.csv")),
                        row.names = 1) 
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaScanpy[deaScanpy$clusters == paste0("cl",cl.val),"clusters"] <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)


  deaScanpy$data_set <- subset.datasets_csv[ind,1]
  deaScanpy$set_number <- ind 
  deaScanpy_tot <- rbind(deaScanpy_tot, deaScanpy)
  
  
  }
 deaScanpy_tot <-  deaScanpy_tot[2:nrow(deaScanpy_tot),]
 
 deaScanpy_tot <- merge.data.frame(deaScanpy_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaScanpy_tot$value <- as.numeric(deaScanpy_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultScanpy <- roc(deaScanpy_tot$value, 1 - deaScanpy_tot$pval)

# Plot the ROC curve
#plot(roc_resultScanpy)

Summary ROC for all methods

TwoClusters_even_near <- ggroc(list(COTAN=roc_resultCOTAN, 
                                    Seurat=roc_resultSeurat,
                 Seurat_scTr = roc_resultSeurat_scTr, 
                 Seurat_Bimod = roc_resultSeurat_Bimod,
                 Monocle=roc_resultMonocle, 
                 Scanpy=roc_resultScanpy),
                 aes = c("colour"),
            legacy.axes = T,
            linewidth = 1)+theme_light()+
  scale_colour_manual("", 
                      values = methods.color)

TwoClusters_even_nearPL <- TwoClusters_even_near + 
  xlab("FPR") + 
  ylab("TPR")
TwoClusters_even_nearPL

2_Clusters_even_medium

True vector

subset.datasets_csv <-datasets_csv[datasets_csv$Group == "2_Clusters_even_medium",] 

ground_truth_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  clusters <- str_split(subset.datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  file.presence.subset <- file.presence[,clusters]
  
  
  #file.presence.subset <- as.matrix(file.presence.subset)
  
  
  ground_truth <- as.data.frame(matrix(nrow = nrow(file.presence.subset),
                                       ncol = ncol(file.presence.subset)))
  rownames(ground_truth) <- rownames(file.presence.subset)
  colnames(ground_truth) <- colnames(file.presence.subset)
  
  ground_truth[file.presence.subset == "Absent"] <- 0
  ground_truth[file.presence.subset == "Present"] <- 1
  ground_truth[file.presence.subset == "Uncertain"] <- 0.6
  
  file.presence.subset <- ground_truth
  
  for (col in 1:ncol(ground_truth)) {
    ground_truth[,col] <- FALSE  
    ground_truth[file.presence.subset[,col] == 1 & rowMeans(file.presence.subset[,-col,drop = FALSE]) < 0.35  ,col] <- TRUE
    }
  
  ground_truth$genes <- rownames(ground_truth) 
  ground_truth <- pivot_longer(ground_truth,
                               cols = 1:(ncol(ground_truth)-1),
                               names_to = "clusters")
  ground_truth$data_set <- subset.datasets_csv[ind,1]
  ground_truth$set_number <- ind 
  ground_truth_tot <- rbind(ground_truth_tot, ground_truth)
  
}
ground_truth_tot <- ground_truth_tot[2:nrow(ground_truth_tot),]

head(ground_truth_tot)
# A tibble: 6 × 5
  genes clusters  value data_set               set_number
  <chr> <chr>     <lgl> <chr>                       <int>
1 Neil2 E13.5-187 FALSE 2_Clusters_even_medium          1
2 Neil2 E13.5-184 FALSE 2_Clusters_even_medium          1
3 Lamc1 E13.5-187 FALSE 2_Clusters_even_medium          1
4 Lamc1 E13.5-184 FALSE 2_Clusters_even_medium          1
5 Lama1 E13.5-187 FALSE 2_Clusters_even_medium          1
6 Lama1 E13.5-184 FALSE 2_Clusters_even_medium          1
length(unique(ground_truth_tot$genes)) 
[1] 14695
sum(ground_truth_tot$value)
[1] 771

ROC for COTAN

onlyPositive.pVal.Cotan_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",
                      subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))
  deaCOTAN <- getClusterizationData(dataset,clName = "mergedClusters")[[2]]
  pvalCOTAN <- pValueFromDEA(deaCOTAN,
              numCells = getNumCells(dataset),method = "none")
  
  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.names <- c(cl.names,
                  str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1])
    cl.names <- cl.names[!is.na(cl.names)]
  }
  colnames(deaCOTAN) <- cl.names
  colnames(pvalCOTAN) <- cl.names
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  onlyPositive.pVal.Cotan <- pvalCOTAN

    for (cl in cl.names) {
    print(cl)
    #temp.DEA.CotanSign <- deaCOTAN[rownames(pvalCOTAN[pvalCOTAN[,cl] < 0.05,]) ,]
    onlyPositive.pVal.Cotan[rownames(deaCOTAN[deaCOTAN[,cl] < 0,]),cl] <-  1 #onlyPositive.pVal.Cotan[rownames(deaCOTAN[deaCOTAN[,cl] < 0,]),cl]+1
    }
    
    onlyPositive.pVal.Cotan$genes <- rownames(onlyPositive.pVal.Cotan) 
    onlyPositive.pVal.Cotan <- pivot_longer(onlyPositive.pVal.Cotan,
                                            values_to = "p_values",
                               cols = 1:(ncol(onlyPositive.pVal.Cotan)-1),
                               names_to = "clusters")
  onlyPositive.pVal.Cotan$data_set <- subset.datasets_csv[ind,1]
  onlyPositive.pVal.Cotan$set_number <- ind 
  onlyPositive.pVal.Cotan_tot <- rbind(onlyPositive.pVal.Cotan_tot, onlyPositive.pVal.Cotan)
  
  }
[1] "E13.5-187"
[1] "E13.5-184"
[1] "E17.5-516"
[1] "E15.0-434"
[1] "E15.0-508"
[1] "E15.0-437"
onlyPositive.pVal.Cotan_tot <- onlyPositive.pVal.Cotan_tot[2:nrow(onlyPositive.pVal.Cotan_tot),]

onlyPositive.pVal.Cotan_tot <- merge.data.frame(onlyPositive.pVal.Cotan_tot,
                                                ground_truth_tot,by = c("genes","clusters","data_set","set_number"),all.x = T,all.y = F)

#onlyPositive.pVal.Cotan_tot <- onlyPositive.pVal.Cotan_tot[order(onlyPositive.pVal.Cotan_tot$p_values,
 #                                                                decreasing = F),]
# df <- as.data.frame(matrix(nrow = nrow(onlyPositive.pVal.Cotan_tot),ncol = 3)) 
# colnames(df) <- c("TPR","FPR","Method")
# df$Method <- "COTAN"
# 
# Positive <- sum(onlyPositive.pVal.Cotan_tot$value) 
# Negative <- sum(!onlyPositive.pVal.Cotan_tot$value)
# 
# for (i in 1:nrow(onlyPositive.pVal.Cotan_tot)) {
#   df[i,"TPR"] <- sum(onlyPositive.pVal.Cotan_tot[1:i,"value"])/Positive
#   df[i,"FPR"] <- (i-sum(onlyPositive.pVal.Cotan_tot[1:i,"value"]))/Negative
  
# Convert TRUE/FALSE to 1/0
onlyPositive.pVal.Cotan_tot$value <- as.numeric(onlyPositive.pVal.Cotan_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultCOTAN <- roc(onlyPositive.pVal.Cotan_tot$value, 1 - onlyPositive.pVal.Cotan_tot$p_values)

# Plot the ROC curve
#plot(roc_resultCOTAN)

ROC for Seurat

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

# Plot the ROC curve
#plot(roc_resultSeurat)

ROC for Seurat scTransform

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_ScTransform_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat_scTr <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

ROC for Seurat Bimod

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_Bimod_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat_Bimod <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

ROC for Monocle

deaMonocle_tot <- NA
for (ind in 1:dim(subset.datasets_csv)[1]) {
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))
  
  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  #print(file.code)
  
  deaMonocle <- read.csv(file.path(dirOut,paste0(file.code,"Monocle_DEA_genes.csv")),row.names = 1) 
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaMonocle[deaMonocle$cell_group == cl.val,"cell_group"] <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaMonocle) <- str_replace(colnames(deaMonocle),
                                     pattern = "cell_group",
                                     replacement = "clusters") 
  colnames(deaMonocle) <- str_replace(colnames(deaMonocle),
                                     pattern = "gene_id",
                                     replacement = "genes")
  
  deaMonocle$data_set <- subset.datasets_csv[ind,1]
  deaMonocle$set_number <- ind 
  deaMonocle <- as.data.frame(deaMonocle)
  deaMonocle_tot <- rbind(deaMonocle_tot, deaMonocle)
  
  
  }
 deaMonocle_tot <-  deaMonocle_tot[2:nrow(deaMonocle_tot),]
 
 deaMonocle_tot <- merge.data.frame(deaMonocle_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaMonocle_tot$value <- as.numeric(deaMonocle_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultMonocle <- roc(deaMonocle_tot$value, 1 - deaMonocle_tot$marker_test_p_value)

# Plot the ROC curve
#plot(roc_resultMonocle)

ROC from Scanpy

deaScanpy_tot <- NA
for (ind in 1:dim(subset.datasets_csv)[1]) {
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  #print(file.code)
  
  deaScanpy <- read.csv(file.path(dirOut,paste0(file.code,"Scanpy_DEA_genes.csv")),
                        row.names = 1) 
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaScanpy[deaScanpy$clusters == paste0("cl",cl.val),"clusters"] <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)


  deaScanpy$data_set <- subset.datasets_csv[ind,1]
  deaScanpy$set_number <- ind 
  deaScanpy_tot <- rbind(deaScanpy_tot, deaScanpy)
  
  
  }
 deaScanpy_tot <-  deaScanpy_tot[2:nrow(deaScanpy_tot),]
 
 deaScanpy_tot <- merge.data.frame(deaScanpy_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaScanpy_tot$value <- as.numeric(deaScanpy_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultScanpy <- roc(deaScanpy_tot$value, 1 - deaScanpy_tot$pval)

# Plot the ROC curve
#plot(roc_resultScanpy)

Summary ROC for all methods

Two_Clusters_even_medium <- ggroc(list(COTAN=roc_resultCOTAN, 
                                    Seurat=roc_resultSeurat,
                 Seurat_scTr = roc_resultSeurat_scTr, 
                 Seurat_Bimod = roc_resultSeurat_Bimod,
                 Monocle=roc_resultMonocle, 
                 Scanpy=roc_resultScanpy),
                 aes = c("colour"),
            legacy.axes = T,
            linewidth = 1)+theme_light()+
  scale_colour_manual("", 
                      values = methods.color)

Two_Clusters_even_mediumPL <- Two_Clusters_even_medium + xlab("FPR") + ylab("TPR")
Two_Clusters_even_mediumPL

2_Clusters_even_far

True vector

subset.datasets_csv <-datasets_csv[datasets_csv$Group == "2_Clusters_even_far",] 

ground_truth_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  clusters <- str_split(subset.datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  file.presence.subset <- file.presence[,clusters]
  
  
  #file.presence.subset <- as.matrix(file.presence.subset)
  
  
  ground_truth <- as.data.frame(matrix(nrow = nrow(file.presence.subset),
                                       ncol = ncol(file.presence.subset)))
  rownames(ground_truth) <- rownames(file.presence.subset)
  colnames(ground_truth) <- colnames(file.presence.subset)
  
  ground_truth[file.presence.subset == "Absent"] <- 0
  ground_truth[file.presence.subset == "Present"] <- 1
  ground_truth[file.presence.subset == "Uncertain"] <- 0.6
  
  file.presence.subset <- ground_truth
  
  for (col in 1:ncol(ground_truth)) {
    ground_truth[,col] <- FALSE  
    ground_truth[file.presence.subset[,col] == 1 & rowMeans(file.presence.subset[,-col,drop = FALSE]) < 0.35  ,col] <- TRUE
    }
  
  ground_truth$genes <- rownames(ground_truth) 
  ground_truth <- pivot_longer(ground_truth,
                               cols = 1:(ncol(ground_truth)-1),
                               names_to = "clusters")
  ground_truth$data_set <- subset.datasets_csv[ind,1]
  ground_truth$set_number <- ind 
  ground_truth_tot <- rbind(ground_truth_tot, ground_truth)
  
}
ground_truth_tot <- ground_truth_tot[2:nrow(ground_truth_tot),]

head(ground_truth_tot)
# A tibble: 6 × 5
  genes clusters  value data_set            set_number
  <chr> <chr>     <lgl> <chr>                    <int>
1 Neil2 E17.5-516 FALSE 2_Clusters_even_far          1
2 Neil2 E13.5-187 FALSE 2_Clusters_even_far          1
3 Lamc1 E17.5-516 FALSE 2_Clusters_even_far          1
4 Lamc1 E13.5-187 FALSE 2_Clusters_even_far          1
5 Lama1 E17.5-516 FALSE 2_Clusters_even_far          1
6 Lama1 E13.5-187 TRUE  2_Clusters_even_far          1
length(unique(ground_truth_tot$genes)) 
[1] 14695
sum(ground_truth_tot$value)
[1] 2325

ROC for COTAN

onlyPositive.pVal.Cotan_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",
                      subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))
  deaCOTAN <- getClusterizationData(dataset,clName = "mergedClusters")[[2]]
  pvalCOTAN <- pValueFromDEA(deaCOTAN,
              numCells = getNumCells(dataset),method = "none")
  
  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.names <- c(cl.names,
                  str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1])
    cl.names <- cl.names[!is.na(cl.names)]
  }
  colnames(deaCOTAN) <- cl.names
  colnames(pvalCOTAN) <- cl.names
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  onlyPositive.pVal.Cotan <- pvalCOTAN

    for (cl in cl.names) {
    print(cl)
    #temp.DEA.CotanSign <- deaCOTAN[rownames(pvalCOTAN[pvalCOTAN[,cl] < 0.05,]) ,]
    onlyPositive.pVal.Cotan[rownames(deaCOTAN[deaCOTAN[,cl] < 0,]),cl] <-  1 #onlyPositive.pVal.Cotan[rownames(deaCOTAN[deaCOTAN[,cl] < 0,]),cl]+1
    }
    
    onlyPositive.pVal.Cotan$genes <- rownames(onlyPositive.pVal.Cotan) 
    onlyPositive.pVal.Cotan <- pivot_longer(onlyPositive.pVal.Cotan,
                                            values_to = "p_values",
                               cols = 1:(ncol(onlyPositive.pVal.Cotan)-1),
                               names_to = "clusters")
  onlyPositive.pVal.Cotan$data_set <- subset.datasets_csv[ind,1]
  onlyPositive.pVal.Cotan$set_number <- ind 
  onlyPositive.pVal.Cotan_tot <- rbind(onlyPositive.pVal.Cotan_tot, onlyPositive.pVal.Cotan)
  
  }
[1] "E13.5-187"
[1] "E17.5-516"
[1] "E15.0-510"
[1] "E13.5-437"
[1] "E15.0-509"
[1] "E13.5-184"
onlyPositive.pVal.Cotan_tot <- onlyPositive.pVal.Cotan_tot[2:nrow(onlyPositive.pVal.Cotan_tot),]

onlyPositive.pVal.Cotan_tot <- merge.data.frame(onlyPositive.pVal.Cotan_tot,
                                                ground_truth_tot,by = c("genes","clusters","data_set","set_number"),all.x = T,all.y = F)

#onlyPositive.pVal.Cotan_tot <- onlyPositive.pVal.Cotan_tot[order(onlyPositive.pVal.Cotan_tot$p_values,
 #                                                                decreasing = F),]
# df <- as.data.frame(matrix(nrow = nrow(onlyPositive.pVal.Cotan_tot),ncol = 3)) 
# colnames(df) <- c("TPR","FPR","Method")
# df$Method <- "COTAN"
# 
# Positive <- sum(onlyPositive.pVal.Cotan_tot$value) 
# Negative <- sum(!onlyPositive.pVal.Cotan_tot$value)
# 
# for (i in 1:nrow(onlyPositive.pVal.Cotan_tot)) {
#   df[i,"TPR"] <- sum(onlyPositive.pVal.Cotan_tot[1:i,"value"])/Positive
#   df[i,"FPR"] <- (i-sum(onlyPositive.pVal.Cotan_tot[1:i,"value"]))/Negative
  
# Convert TRUE/FALSE to 1/0
onlyPositive.pVal.Cotan_tot$value <- as.numeric(onlyPositive.pVal.Cotan_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultCOTAN <- roc(onlyPositive.pVal.Cotan_tot$value, 1 - onlyPositive.pVal.Cotan_tot$p_values)

# Plot the ROC curve
#plot(roc_resultCOTAN)

ROC for Seurat

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

# Plot the ROC curve
#plot(roc_resultSeurat)

ROC for Seurat scTransform

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_ScTransform_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat_scTr <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

ROC for Seurat Bimod

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_Bimod_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat_Bimod <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

ROC for Monocle

deaMonocle_tot <- NA
for (ind in 1:dim(subset.datasets_csv)[1]) {
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))
  
  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  #print(file.code)
  
  deaMonocle <- read.csv(file.path(dirOut,paste0(file.code,"Monocle_DEA_genes.csv")),row.names = 1) 
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaMonocle[deaMonocle$cell_group == cl.val,"cell_group"] <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaMonocle) <- str_replace(colnames(deaMonocle),
                                     pattern = "cell_group",
                                     replacement = "clusters") 
  colnames(deaMonocle) <- str_replace(colnames(deaMonocle),
                                     pattern = "gene_id",
                                     replacement = "genes")
  
  deaMonocle$data_set <- subset.datasets_csv[ind,1]
  deaMonocle$set_number <- ind 
  deaMonocle <- as.data.frame(deaMonocle)
  deaMonocle_tot <- rbind(deaMonocle_tot, deaMonocle)
  
  
  }
 deaMonocle_tot <-  deaMonocle_tot[2:nrow(deaMonocle_tot),]
 
 deaMonocle_tot <- merge.data.frame(deaMonocle_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaMonocle_tot$value <- as.numeric(deaMonocle_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultMonocle <- roc(deaMonocle_tot$value, 1 - deaMonocle_tot$marker_test_p_value)

# Plot the ROC curve
#plot(roc_resultMonocle)

ROC from Scanpy

deaScanpy_tot <- NA
for (ind in 1:dim(subset.datasets_csv)[1]) {
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  #print(file.code)
  
  deaScanpy <- read.csv(file.path(dirOut,paste0(file.code,"Scanpy_DEA_genes.csv")),
                        row.names = 1) 
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaScanpy[deaScanpy$clusters == paste0("cl",cl.val),"clusters"] <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)


  deaScanpy$data_set <- subset.datasets_csv[ind,1]
  deaScanpy$set_number <- ind 
  deaScanpy_tot <- rbind(deaScanpy_tot, deaScanpy)
  
  
  }
 deaScanpy_tot <-  deaScanpy_tot[2:nrow(deaScanpy_tot),]
 
 deaScanpy_tot <- merge.data.frame(deaScanpy_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaScanpy_tot$value <- as.numeric(deaScanpy_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultScanpy <- roc(deaScanpy_tot$value, 1 - deaScanpy_tot$pval)

# Plot the ROC curve
#plot(roc_resultScanpy)

Summary ROC for all methods

Two_Clusters_even_far <- ggroc(list(COTAN=roc_resultCOTAN, 
                                    Seurat=roc_resultSeurat,
                 Seurat_scTr = roc_resultSeurat_scTr, 
                 Seurat_Bimod = roc_resultSeurat_Bimod,
                 Monocle=roc_resultMonocle, 
                 Scanpy=roc_resultScanpy),
                 aes = c("colour"),
            legacy.axes = T,
            linewidth = 1)+theme_light()+
  scale_colour_manual("", 
                      values = methods.color)

Two_Clusters_even_farPL <- Two_Clusters_even_far + xlab("FPR") + ylab("TPR")
Two_Clusters_even_farPL

2 clusters uneven

2_Clusters_uneven_near

True vector

subset.datasets_csv <-datasets_csv[datasets_csv$Group == "2_Clusters_uneven_near",] 

ground_truth_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  clusters <- str_split(subset.datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  file.presence.subset <- file.presence[,clusters]
  
  
  #file.presence.subset <- as.matrix(file.presence.subset)
  
  
  ground_truth <- as.data.frame(matrix(nrow = nrow(file.presence.subset),
                                       ncol = ncol(file.presence.subset)))
  rownames(ground_truth) <- rownames(file.presence.subset)
  colnames(ground_truth) <- colnames(file.presence.subset)
  
  ground_truth[file.presence.subset == "Absent"] <- 0
  ground_truth[file.presence.subset == "Present"] <- 1
  ground_truth[file.presence.subset == "Uncertain"] <- 0.6
  
  file.presence.subset <- ground_truth
  
  for (col in 1:ncol(ground_truth)) {
    ground_truth[,col] <- FALSE  
    ground_truth[file.presence.subset[,col] == 1 & rowMeans(file.presence.subset[,-col,drop = FALSE]) < 0.35  ,col] <- TRUE
    }
  
  ground_truth$genes <- rownames(ground_truth) 
  ground_truth <- pivot_longer(ground_truth,
                               cols = 1:(ncol(ground_truth)-1),
                               names_to = "clusters")
  ground_truth$data_set <- subset.datasets_csv[ind,1]
  ground_truth$set_number <- ind 
  ground_truth_tot <- rbind(ground_truth_tot, ground_truth)
  
}
ground_truth_tot <- ground_truth_tot[2:nrow(ground_truth_tot),]

head(ground_truth_tot)
# A tibble: 6 × 5
  genes clusters  value data_set               set_number
  <chr> <chr>     <lgl> <chr>                       <int>
1 Neil2 E13.5-434 FALSE 2_Clusters_uneven_near          1
2 Neil2 E15.0-428 FALSE 2_Clusters_uneven_near          1
3 Lamc1 E13.5-434 FALSE 2_Clusters_uneven_near          1
4 Lamc1 E15.0-428 FALSE 2_Clusters_uneven_near          1
5 Lama1 E13.5-434 FALSE 2_Clusters_uneven_near          1
6 Lama1 E15.0-428 FALSE 2_Clusters_uneven_near          1
length(unique(ground_truth_tot$genes)) 
[1] 14695
sum(ground_truth_tot$value)
[1] 24

ROC for COTAN

onlyPositive.pVal.Cotan_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",
                      subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))
  deaCOTAN <- getClusterizationData(dataset,clName = "mergedClusters")[[2]]
  pvalCOTAN <- pValueFromDEA(deaCOTAN,
              numCells = getNumCells(dataset),method = "none")
  
  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.names <- c(cl.names,
                  str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1])
    cl.names <- cl.names[!is.na(cl.names)]
  }
  colnames(deaCOTAN) <- cl.names
  colnames(pvalCOTAN) <- cl.names
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  onlyPositive.pVal.Cotan <- pvalCOTAN

    for (cl in cl.names) {
    print(cl)
    #temp.DEA.CotanSign <- deaCOTAN[rownames(pvalCOTAN[pvalCOTAN[,cl] < 0.05,]) ,]
    onlyPositive.pVal.Cotan[rownames(deaCOTAN[deaCOTAN[,cl] < 0,]),cl] <-  1 #onlyPositive.pVal.Cotan[rownames(deaCOTAN[deaCOTAN[,cl] < 0,]),cl]+1
    }
    
    onlyPositive.pVal.Cotan$genes <- rownames(onlyPositive.pVal.Cotan) 
    onlyPositive.pVal.Cotan <- pivot_longer(onlyPositive.pVal.Cotan,
                                            values_to = "p_values",
                               cols = 1:(ncol(onlyPositive.pVal.Cotan)-1),
                               names_to = "clusters")
  onlyPositive.pVal.Cotan$data_set <- subset.datasets_csv[ind,1]
  onlyPositive.pVal.Cotan$set_number <- ind 
  onlyPositive.pVal.Cotan_tot <- rbind(onlyPositive.pVal.Cotan_tot, onlyPositive.pVal.Cotan)
  
  }
[1] "E13.5-434"
[1] "E15.0-428"
[1] "E15.0-432"
[1] "E13.5-432"
[1] "E15.0-509"
[1] "E15.0-508"
onlyPositive.pVal.Cotan_tot <- onlyPositive.pVal.Cotan_tot[2:nrow(onlyPositive.pVal.Cotan_tot),]

onlyPositive.pVal.Cotan_tot <- merge.data.frame(onlyPositive.pVal.Cotan_tot,
                                                ground_truth_tot,by = c("genes","clusters","data_set","set_number"),all.x = T,all.y = F)

#onlyPositive.pVal.Cotan_tot <- onlyPositive.pVal.Cotan_tot[order(onlyPositive.pVal.Cotan_tot$p_values,
 #                                                                decreasing = F),]
# df <- as.data.frame(matrix(nrow = nrow(onlyPositive.pVal.Cotan_tot),ncol = 3)) 
# colnames(df) <- c("TPR","FPR","Method")
# df$Method <- "COTAN"
# 
# Positive <- sum(onlyPositive.pVal.Cotan_tot$value) 
# Negative <- sum(!onlyPositive.pVal.Cotan_tot$value)
# 
# for (i in 1:nrow(onlyPositive.pVal.Cotan_tot)) {
#   df[i,"TPR"] <- sum(onlyPositive.pVal.Cotan_tot[1:i,"value"])/Positive
#   df[i,"FPR"] <- (i-sum(onlyPositive.pVal.Cotan_tot[1:i,"value"]))/Negative
  
# Convert TRUE/FALSE to 1/0
onlyPositive.pVal.Cotan_tot$value <- as.numeric(onlyPositive.pVal.Cotan_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultCOTAN <- roc(onlyPositive.pVal.Cotan_tot$value, 1 - onlyPositive.pVal.Cotan_tot$p_values)

# Plot the ROC curve
#plot(roc_resultCOTAN)

ROC for Seurat

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

# Plot the ROC curve
plot(roc_resultSeurat)

ROC for Seurat scTransform

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_ScTransform_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat_scTr <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

ROC for Seurat Bimod

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_Bimod_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat_Bimod <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

ROC for Monocle

deaMonocle_tot <- NA
for (ind in 1:dim(subset.datasets_csv)[1]) {
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))
  
  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  #print(file.code)
  
  deaMonocle <- read.csv(file.path(dirOut,paste0(file.code,"Monocle_DEA_genes.csv")),row.names = 1) 
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaMonocle[deaMonocle$cell_group == cl.val,"cell_group"] <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaMonocle) <- str_replace(colnames(deaMonocle),
                                     pattern = "cell_group",
                                     replacement = "clusters") 
  colnames(deaMonocle) <- str_replace(colnames(deaMonocle),
                                     pattern = "gene_id",
                                     replacement = "genes")
  
  deaMonocle$data_set <- subset.datasets_csv[ind,1]
  deaMonocle$set_number <- ind 
  deaMonocle <- as.data.frame(deaMonocle)
  deaMonocle_tot <- rbind(deaMonocle_tot, deaMonocle)
  
  
  }
 deaMonocle_tot <-  deaMonocle_tot[2:nrow(deaMonocle_tot),]
 
 deaMonocle_tot <- merge.data.frame(deaMonocle_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaMonocle_tot$value <- as.numeric(deaMonocle_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultMonocle <- roc(deaMonocle_tot$value, 1 - deaMonocle_tot$marker_test_p_value)

# Plot the ROC curve
#plot(roc_resultMonocle)

ROC from Scanpy

deaScanpy_tot <- NA
for (ind in 1:dim(subset.datasets_csv)[1]) {
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  #print(file.code)
  
  deaScanpy <- read.csv(file.path(dirOut,paste0(file.code,"Scanpy_DEA_genes.csv")),
                        row.names = 1) 
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaScanpy[deaScanpy$clusters == paste0("cl",cl.val),"clusters"] <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)


  deaScanpy$data_set <- subset.datasets_csv[ind,1]
  deaScanpy$set_number <- ind 
  deaScanpy_tot <- rbind(deaScanpy_tot, deaScanpy)
  
  
  }
 deaScanpy_tot <-  deaScanpy_tot[2:nrow(deaScanpy_tot),]
 
 deaScanpy_tot <- merge.data.frame(deaScanpy_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaScanpy_tot$value <- as.numeric(deaScanpy_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultScanpy <- roc(deaScanpy_tot$value, 1 - deaScanpy_tot$pval)

# Plot the ROC curve
#plot(roc_resultScanpy)

Summary ROC for all methods

Two_Clusters_uneven_near <- ggroc(list(COTAN=roc_resultCOTAN, 
                                    Seurat=roc_resultSeurat,
                 Seurat_scTr = roc_resultSeurat_scTr, 
                 Seurat_Bimod = roc_resultSeurat_Bimod,
                 Monocle=roc_resultMonocle, 
                 Scanpy=roc_resultScanpy),
                 aes = c("colour"),
            legacy.axes = T,
            linewidth = 1)+theme_light()+
  scale_colour_manual("", 
                      values = methods.color)

Two_Clusters_uneven_nearPL <- Two_Clusters_uneven_near + xlab("FPR") + ylab("TPR")
Two_Clusters_uneven_nearPL

2_Clusters_uneven_medium

True vector

subset.datasets_csv <-datasets_csv[datasets_csv$Group == "2_Clusters_uneven_medium",] 

ground_truth_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  clusters <- str_split(subset.datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  file.presence.subset <- file.presence[,clusters]
  
  
  #file.presence.subset <- as.matrix(file.presence.subset)
  
  
  ground_truth <- as.data.frame(matrix(nrow = nrow(file.presence.subset),
                                       ncol = ncol(file.presence.subset)))
  rownames(ground_truth) <- rownames(file.presence.subset)
  colnames(ground_truth) <- colnames(file.presence.subset)
  
  ground_truth[file.presence.subset == "Absent"] <- 0
  ground_truth[file.presence.subset == "Present"] <- 1
  ground_truth[file.presence.subset == "Uncertain"] <- 0.6
  
  file.presence.subset <- ground_truth
  
  for (col in 1:ncol(ground_truth)) {
    ground_truth[,col] <- FALSE  
    ground_truth[file.presence.subset[,col] == 1 & rowMeans(file.presence.subset[,-col,drop = FALSE]) < 0.35  ,col] <- TRUE
    }
  
  ground_truth$genes <- rownames(ground_truth) 
  ground_truth <- pivot_longer(ground_truth,
                               cols = 1:(ncol(ground_truth)-1),
                               names_to = "clusters")
  ground_truth$data_set <- subset.datasets_csv[ind,1]
  ground_truth$set_number <- ind 
  ground_truth_tot <- rbind(ground_truth_tot, ground_truth)
  
}
ground_truth_tot <- ground_truth_tot[2:nrow(ground_truth_tot),]

head(ground_truth_tot)
# A tibble: 6 × 5
  genes clusters  value data_set                 set_number
  <chr> <chr>     <lgl> <chr>                         <int>
1 Neil2 E13.5-187 FALSE 2_Clusters_uneven_medium          1
2 Neil2 E13.5-184 FALSE 2_Clusters_uneven_medium          1
3 Lamc1 E13.5-187 FALSE 2_Clusters_uneven_medium          1
4 Lamc1 E13.5-184 FALSE 2_Clusters_uneven_medium          1
5 Lama1 E13.5-187 FALSE 2_Clusters_uneven_medium          1
6 Lama1 E13.5-184 FALSE 2_Clusters_uneven_medium          1
length(unique(ground_truth_tot$genes)) 
[1] 14695
sum(ground_truth_tot$value)
[1] 771

ROC for COTAN

onlyPositive.pVal.Cotan_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",
                      subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))
  deaCOTAN <- getClusterizationData(dataset,clName = "mergedClusters")[[2]]
  pvalCOTAN <- pValueFromDEA(deaCOTAN,
              numCells = getNumCells(dataset),method = "none")
  
  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.names <- c(cl.names,
                  str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1])
    cl.names <- cl.names[!is.na(cl.names)]
  }
  colnames(deaCOTAN) <- cl.names
  colnames(pvalCOTAN) <- cl.names
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  onlyPositive.pVal.Cotan <- pvalCOTAN

    for (cl in cl.names) {
    print(cl)
    #temp.DEA.CotanSign <- deaCOTAN[rownames(pvalCOTAN[pvalCOTAN[,cl] < 0.05,]) ,]
    onlyPositive.pVal.Cotan[rownames(deaCOTAN[deaCOTAN[,cl] < 0,]),cl] <-  1 #onlyPositive.pVal.Cotan[rownames(deaCOTAN[deaCOTAN[,cl] < 0,]),cl]+1
    }
    
    onlyPositive.pVal.Cotan$genes <- rownames(onlyPositive.pVal.Cotan) 
    onlyPositive.pVal.Cotan <- pivot_longer(onlyPositive.pVal.Cotan,
                                            values_to = "p_values",
                               cols = 1:(ncol(onlyPositive.pVal.Cotan)-1),
                               names_to = "clusters")
  onlyPositive.pVal.Cotan$data_set <- subset.datasets_csv[ind,1]
  onlyPositive.pVal.Cotan$set_number <- ind 
  onlyPositive.pVal.Cotan_tot <- rbind(onlyPositive.pVal.Cotan_tot, onlyPositive.pVal.Cotan)
  
  }
[1] "E13.5-187"
[1] "E13.5-184"
[1] "E17.5-516"
[1] "E15.0-434"
[1] "E15.0-508"
[1] "E15.0-437"
onlyPositive.pVal.Cotan_tot <- onlyPositive.pVal.Cotan_tot[2:nrow(onlyPositive.pVal.Cotan_tot),]

onlyPositive.pVal.Cotan_tot <- merge.data.frame(onlyPositive.pVal.Cotan_tot,
                                                ground_truth_tot,by = c("genes","clusters","data_set","set_number"),all.x = T,all.y = F)

#onlyPositive.pVal.Cotan_tot <- onlyPositive.pVal.Cotan_tot[order(onlyPositive.pVal.Cotan_tot$p_values,
 #                                                                decreasing = F),]
# df <- as.data.frame(matrix(nrow = nrow(onlyPositive.pVal.Cotan_tot),ncol = 3)) 
# colnames(df) <- c("TPR","FPR","Method")
# df$Method <- "COTAN"
# 
# Positive <- sum(onlyPositive.pVal.Cotan_tot$value) 
# Negative <- sum(!onlyPositive.pVal.Cotan_tot$value)
# 
# for (i in 1:nrow(onlyPositive.pVal.Cotan_tot)) {
#   df[i,"TPR"] <- sum(onlyPositive.pVal.Cotan_tot[1:i,"value"])/Positive
#   df[i,"FPR"] <- (i-sum(onlyPositive.pVal.Cotan_tot[1:i,"value"]))/Negative
  
# Convert TRUE/FALSE to 1/0
onlyPositive.pVal.Cotan_tot$value <- as.numeric(onlyPositive.pVal.Cotan_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultCOTAN <- roc(onlyPositive.pVal.Cotan_tot$value, 1 - onlyPositive.pVal.Cotan_tot$p_values)

# Plot the ROC curve
#plot(roc_resultCOTAN)

ROC for Seurat

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

# Plot the ROC curve
#plot(roc_resultSeurat)

ROC for Seurat scTransform

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_ScTransform_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat_scTr <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

ROC for Seurat Bimod

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_Bimod_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat_Bimod <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

ROC for Monocle

deaMonocle_tot <- NA
for (ind in 1:dim(subset.datasets_csv)[1]) {
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))
  
  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  #print(file.code)
  
  deaMonocle <- read.csv(file.path(dirOut,paste0(file.code,"Monocle_DEA_genes.csv")),row.names = 1) 
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaMonocle[deaMonocle$cell_group == cl.val,"cell_group"] <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaMonocle) <- str_replace(colnames(deaMonocle),
                                     pattern = "cell_group",
                                     replacement = "clusters") 
  colnames(deaMonocle) <- str_replace(colnames(deaMonocle),
                                     pattern = "gene_id",
                                     replacement = "genes")
  
  deaMonocle$data_set <- subset.datasets_csv[ind,1]
  deaMonocle$set_number <- ind 
  deaMonocle <- as.data.frame(deaMonocle)
  deaMonocle_tot <- rbind(deaMonocle_tot, deaMonocle)
  
  
  }
 deaMonocle_tot <-  deaMonocle_tot[2:nrow(deaMonocle_tot),]
 
 deaMonocle_tot <- merge.data.frame(deaMonocle_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaMonocle_tot$value <- as.numeric(deaMonocle_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultMonocle <- roc(deaMonocle_tot$value, 1 - deaMonocle_tot$marker_test_p_value)

# Plot the ROC curve
#plot(roc_resultMonocle)

ROC from Scanpy

deaScanpy_tot <- NA
for (ind in 1:dim(subset.datasets_csv)[1]) {
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  #print(file.code)
  
  deaScanpy <- read.csv(file.path(dirOut,paste0(file.code,"Scanpy_DEA_genes.csv")),
                        row.names = 1) 
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaScanpy[deaScanpy$clusters == paste0("cl",cl.val),"clusters"] <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)


  deaScanpy$data_set <- subset.datasets_csv[ind,1]
  deaScanpy$set_number <- ind 
  deaScanpy_tot <- rbind(deaScanpy_tot, deaScanpy)
  
  
  }
 deaScanpy_tot <-  deaScanpy_tot[2:nrow(deaScanpy_tot),]
 
 deaScanpy_tot <- merge.data.frame(deaScanpy_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaScanpy_tot$value <- as.numeric(deaScanpy_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultScanpy <- roc(deaScanpy_tot$value, 1 - deaScanpy_tot$pval)

# Plot the ROC curve
#plot(roc_resultScanpy)

Summary ROC for all methods

Two_Clusters_uneven_medium <- ggroc(list(COTAN=roc_resultCOTAN, 
                                    Seurat=roc_resultSeurat,
                 Seurat_scTr = roc_resultSeurat_scTr, 
                 Seurat_Bimod = roc_resultSeurat_Bimod,
                 Monocle=roc_resultMonocle, 
                 Scanpy=roc_resultScanpy),
                 aes = c("colour"),
            legacy.axes = T,
            linewidth = 1)+theme_light()+
  scale_colour_manual("", 
                      values = methods.color)

Two_Clusters_uneven_mediumPL <- Two_Clusters_uneven_medium + xlab("FPR") + ylab("TPR")

Two_Clusters_uneven_mediumPL

2_Clusters_uneven_far

True vector

subset.datasets_csv <-datasets_csv[datasets_csv$Group == "2_Clusters_uneven_far",] 

ground_truth_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  clusters <- str_split(subset.datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  file.presence.subset <- file.presence[,clusters]
  
  
  #file.presence.subset <- as.matrix(file.presence.subset)
  
  
  ground_truth <- as.data.frame(matrix(nrow = nrow(file.presence.subset),
                                       ncol = ncol(file.presence.subset)))
  rownames(ground_truth) <- rownames(file.presence.subset)
  colnames(ground_truth) <- colnames(file.presence.subset)
  
  ground_truth[file.presence.subset == "Absent"] <- 0
  ground_truth[file.presence.subset == "Present"] <- 1
  ground_truth[file.presence.subset == "Uncertain"] <- 0.6
  
  file.presence.subset <- ground_truth
  
  for (col in 1:ncol(ground_truth)) {
    ground_truth[,col] <- FALSE  
    ground_truth[file.presence.subset[,col] == 1 & rowMeans(file.presence.subset[,-col,drop = FALSE]) < 0.35  ,col] <- TRUE
    }
  
  ground_truth$genes <- rownames(ground_truth) 
  ground_truth <- pivot_longer(ground_truth,
                               cols = 1:(ncol(ground_truth)-1),
                               names_to = "clusters")
  ground_truth$data_set <- subset.datasets_csv[ind,1]
  ground_truth$set_number <- ind 
  ground_truth_tot <- rbind(ground_truth_tot, ground_truth)
  
}
ground_truth_tot <- ground_truth_tot[2:nrow(ground_truth_tot),]

head(ground_truth_tot)
# A tibble: 6 × 5
  genes clusters  value data_set              set_number
  <chr> <chr>     <lgl> <chr>                      <int>
1 Neil2 E17.5-516 FALSE 2_Clusters_uneven_far          1
2 Neil2 E13.5-187 FALSE 2_Clusters_uneven_far          1
3 Lamc1 E17.5-516 FALSE 2_Clusters_uneven_far          1
4 Lamc1 E13.5-187 FALSE 2_Clusters_uneven_far          1
5 Lama1 E17.5-516 FALSE 2_Clusters_uneven_far          1
6 Lama1 E13.5-187 TRUE  2_Clusters_uneven_far          1
length(unique(ground_truth_tot$genes)) 
[1] 14695
sum(ground_truth_tot$value)
[1] 2325

ROC for COTAN

onlyPositive.pVal.Cotan_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",
                      subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))
  deaCOTAN <- getClusterizationData(dataset,clName = "mergedClusters")[[2]]
  pvalCOTAN <- pValueFromDEA(deaCOTAN,
              numCells = getNumCells(dataset),method = "none")
  
  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.names <- c(cl.names,
                  str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1])
    cl.names <- cl.names[!is.na(cl.names)]
  }
  colnames(deaCOTAN) <- cl.names
  colnames(pvalCOTAN) <- cl.names
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  onlyPositive.pVal.Cotan <- pvalCOTAN

    for (cl in cl.names) {
    print(cl)
    #temp.DEA.CotanSign <- deaCOTAN[rownames(pvalCOTAN[pvalCOTAN[,cl] < 0.05,]) ,]
    onlyPositive.pVal.Cotan[rownames(deaCOTAN[deaCOTAN[,cl] < 0,]),cl] <-  1 #onlyPositive.pVal.Cotan[rownames(deaCOTAN[deaCOTAN[,cl] < 0,]),cl]+1
    }
    
    onlyPositive.pVal.Cotan$genes <- rownames(onlyPositive.pVal.Cotan) 
    onlyPositive.pVal.Cotan <- pivot_longer(onlyPositive.pVal.Cotan,
                                            values_to = "p_values",
                               cols = 1:(ncol(onlyPositive.pVal.Cotan)-1),
                               names_to = "clusters")
  onlyPositive.pVal.Cotan$data_set <- subset.datasets_csv[ind,1]
  onlyPositive.pVal.Cotan$set_number <- ind 
  onlyPositive.pVal.Cotan_tot <- rbind(onlyPositive.pVal.Cotan_tot, onlyPositive.pVal.Cotan)
  
  }
[1] "E13.5-187"
[1] "E17.5-516"
[1] "E15.0-510"
[1] "E13.5-437"
[1] "E15.0-509"
[1] "E13.5-184"
onlyPositive.pVal.Cotan_tot <- onlyPositive.pVal.Cotan_tot[2:nrow(onlyPositive.pVal.Cotan_tot),]

onlyPositive.pVal.Cotan_tot <- merge.data.frame(onlyPositive.pVal.Cotan_tot,
                                                ground_truth_tot,by = c("genes","clusters","data_set","set_number"),all.x = T,all.y = F)

#onlyPositive.pVal.Cotan_tot <- onlyPositive.pVal.Cotan_tot[order(onlyPositive.pVal.Cotan_tot$p_values,
 #                                                                decreasing = F),]
# df <- as.data.frame(matrix(nrow = nrow(onlyPositive.pVal.Cotan_tot),ncol = 3)) 
# colnames(df) <- c("TPR","FPR","Method")
# df$Method <- "COTAN"
# 
# Positive <- sum(onlyPositive.pVal.Cotan_tot$value) 
# Negative <- sum(!onlyPositive.pVal.Cotan_tot$value)
# 
# for (i in 1:nrow(onlyPositive.pVal.Cotan_tot)) {
#   df[i,"TPR"] <- sum(onlyPositive.pVal.Cotan_tot[1:i,"value"])/Positive
#   df[i,"FPR"] <- (i-sum(onlyPositive.pVal.Cotan_tot[1:i,"value"]))/Negative
  
# Convert TRUE/FALSE to 1/0
onlyPositive.pVal.Cotan_tot$value <- as.numeric(onlyPositive.pVal.Cotan_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultCOTAN <- roc(onlyPositive.pVal.Cotan_tot$value, 1 - onlyPositive.pVal.Cotan_tot$p_values)

# Plot the ROC curve
#plot(roc_resultCOTAN)

ROC for Seurat

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

# Plot the ROC curve
#plot(roc_resultSeurat)

ROC for Seurat scTransform

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_ScTransform_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat_scTr <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

ROC for Seurat Bimod

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_Bimod_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat_Bimod <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

ROC for Monocle

deaMonocle_tot <- NA
for (ind in 1:dim(subset.datasets_csv)[1]) {
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))
  
  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  #print(file.code)
  
  deaMonocle <- read.csv(file.path(dirOut,paste0(file.code,"Monocle_DEA_genes.csv")),row.names = 1) 
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaMonocle[deaMonocle$cell_group == cl.val,"cell_group"] <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaMonocle) <- str_replace(colnames(deaMonocle),
                                     pattern = "cell_group",
                                     replacement = "clusters") 
  colnames(deaMonocle) <- str_replace(colnames(deaMonocle),
                                     pattern = "gene_id",
                                     replacement = "genes")
  
  deaMonocle$data_set <- subset.datasets_csv[ind,1]
  deaMonocle$set_number <- ind 
  deaMonocle <- as.data.frame(deaMonocle)
  deaMonocle_tot <- rbind(deaMonocle_tot, deaMonocle)
  
  
  }
 deaMonocle_tot <-  deaMonocle_tot[2:nrow(deaMonocle_tot),]
 
 deaMonocle_tot <- merge.data.frame(deaMonocle_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaMonocle_tot$value <- as.numeric(deaMonocle_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultMonocle <- roc(deaMonocle_tot$value, 1 - deaMonocle_tot$marker_test_p_value)

# Plot the ROC curve
#plot(roc_resultMonocle)

ROC from Scanpy

deaScanpy_tot <- NA
for (ind in 1:dim(subset.datasets_csv)[1]) {
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  #print(file.code)
  
  deaScanpy <- read.csv(file.path(dirOut,paste0(file.code,"Scanpy_DEA_genes.csv")),
                        row.names = 1) 
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaScanpy[deaScanpy$clusters == paste0("cl",cl.val),"clusters"] <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)


  deaScanpy$data_set <- subset.datasets_csv[ind,1]
  deaScanpy$set_number <- ind 
  deaScanpy_tot <- rbind(deaScanpy_tot, deaScanpy)
  
  
  }
 deaScanpy_tot <-  deaScanpy_tot[2:nrow(deaScanpy_tot),]
 
 deaScanpy_tot <- merge.data.frame(deaScanpy_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaScanpy_tot$value <- as.numeric(deaScanpy_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultScanpy <- roc(deaScanpy_tot$value, 1 - deaScanpy_tot$pval)

# Plot the ROC curve
#plot(roc_resultScanpy)

Summary ROC for all methods

Two_Clusters_uneven_far <- ggroc(list(COTAN=roc_resultCOTAN, 
                                    Seurat=roc_resultSeurat,
                 Seurat_scTr = roc_resultSeurat_scTr, 
                 Seurat_Bimod = roc_resultSeurat_Bimod,
                 Monocle=roc_resultMonocle, 
                 Scanpy=roc_resultScanpy),
                 aes = c("colour"),
            legacy.axes = T,
            linewidth = 1)+theme_light()+
  scale_colour_manual("", 
                      values = methods.color)

Two_Clusters_uneven_farPL <- Two_Clusters_uneven_far + xlab("FPR") + ylab("TPR")

lg <- get_legend(Two_Clusters_uneven_farPL)

Two_Clusters_uneven_farPL

3 clusters

3_Clusters_even

True vector

subset.datasets_csv <-datasets_csv[datasets_csv$Group == "3_Clusters_even",] 

ground_truth_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  clusters <- str_split(subset.datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  file.presence.subset <- file.presence[,clusters]
  
  
  #file.presence.subset <- as.matrix(file.presence.subset)
  
  
  ground_truth <- as.data.frame(matrix(nrow = nrow(file.presence.subset),
                                       ncol = ncol(file.presence.subset)))
  rownames(ground_truth) <- rownames(file.presence.subset)
  colnames(ground_truth) <- colnames(file.presence.subset)
  
  ground_truth[file.presence.subset == "Absent"] <- 0
  ground_truth[file.presence.subset == "Present"] <- 1
  ground_truth[file.presence.subset == "Uncertain"] <- 0.6
  
  file.presence.subset <- ground_truth
  
  for (col in 1:ncol(ground_truth)) {
    ground_truth[,col] <- FALSE  
    ground_truth[file.presence.subset[,col] == 1 & rowMeans(file.presence.subset[,-col,drop = FALSE]) < 0.35  ,col] <- TRUE
    }
  
  ground_truth$genes <- rownames(ground_truth) 
  ground_truth <- pivot_longer(ground_truth,
                               cols = 1:(ncol(ground_truth)-1),
                               names_to = "clusters")
  ground_truth$data_set <- subset.datasets_csv[ind,1]
  ground_truth$set_number <- ind 
  ground_truth_tot <- rbind(ground_truth_tot, ground_truth)
  
}
ground_truth_tot <- ground_truth_tot[2:nrow(ground_truth_tot),]

head(ground_truth_tot)
# A tibble: 6 × 5
  genes clusters  value data_set        set_number
  <chr> <chr>     <lgl> <chr>                <int>
1 Neil2 E15.0-437 FALSE 3_Clusters_even          1
2 Neil2 E13.5-510 FALSE 3_Clusters_even          1
3 Neil2 E13.5-437 FALSE 3_Clusters_even          1
4 Lamc1 E15.0-437 FALSE 3_Clusters_even          1
5 Lamc1 E13.5-510 FALSE 3_Clusters_even          1
6 Lamc1 E13.5-437 FALSE 3_Clusters_even          1
length(unique(ground_truth_tot$genes)) 
[1] 14695
sum(ground_truth_tot$value)
[1] 1288

ROC for COTAN

onlyPositive.pVal.Cotan_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",
                      subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))
  deaCOTAN <- getClusterizationData(dataset,clName = "mergedClusters")[[2]]
  pvalCOTAN <- pValueFromDEA(deaCOTAN,
              numCells = getNumCells(dataset),method = "none")
  
  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.names <- c(cl.names,
                  str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1])
    cl.names <- cl.names[!is.na(cl.names)]
  }
  colnames(deaCOTAN) <- cl.names
  colnames(pvalCOTAN) <- cl.names
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  onlyPositive.pVal.Cotan <- pvalCOTAN

    for (cl in cl.names) {
    print(cl)
    #temp.DEA.CotanSign <- deaCOTAN[rownames(pvalCOTAN[pvalCOTAN[,cl] < 0.05,]) ,]
    onlyPositive.pVal.Cotan[rownames(deaCOTAN[deaCOTAN[,cl] < 0,]),cl] <-  1 #onlyPositive.pVal.Cotan[rownames(deaCOTAN[deaCOTAN[,cl] < 0,]),cl]+1
    }
    
    onlyPositive.pVal.Cotan$genes <- rownames(onlyPositive.pVal.Cotan) 
    onlyPositive.pVal.Cotan <- pivot_longer(onlyPositive.pVal.Cotan,
                                            values_to = "p_values",
                               cols = 1:(ncol(onlyPositive.pVal.Cotan)-1),
                               names_to = "clusters")
  onlyPositive.pVal.Cotan$data_set <- subset.datasets_csv[ind,1]
  onlyPositive.pVal.Cotan$set_number <- ind 
  onlyPositive.pVal.Cotan_tot <- rbind(onlyPositive.pVal.Cotan_tot, onlyPositive.pVal.Cotan)
  
  }
[1] "E13.5-437"
[1] "E15.0-437"
[1] "E13.5-510"
[1] "E17.5-516"
[1] "E13.5-437"
[1] "E17.5-505"
[1] "E15.0-510"
[1] "E15.0-428"
[1] "E13.5-510"
onlyPositive.pVal.Cotan_tot <- onlyPositive.pVal.Cotan_tot[2:nrow(onlyPositive.pVal.Cotan_tot),]

onlyPositive.pVal.Cotan_tot <- merge.data.frame(onlyPositive.pVal.Cotan_tot,
                                                ground_truth_tot,by = c("genes","clusters","data_set","set_number"),all.x = T,all.y = F)

#onlyPositive.pVal.Cotan_tot <- onlyPositive.pVal.Cotan_tot[order(onlyPositive.pVal.Cotan_tot$p_values,
 #                                                                decreasing = F),]
# df <- as.data.frame(matrix(nrow = nrow(onlyPositive.pVal.Cotan_tot),ncol = 3)) 
# colnames(df) <- c("TPR","FPR","Method")
# df$Method <- "COTAN"
# 
# Positive <- sum(onlyPositive.pVal.Cotan_tot$value) 
# Negative <- sum(!onlyPositive.pVal.Cotan_tot$value)
# 
# for (i in 1:nrow(onlyPositive.pVal.Cotan_tot)) {
#   df[i,"TPR"] <- sum(onlyPositive.pVal.Cotan_tot[1:i,"value"])/Positive
#   df[i,"FPR"] <- (i-sum(onlyPositive.pVal.Cotan_tot[1:i,"value"]))/Negative
  
# Convert TRUE/FALSE to 1/0
onlyPositive.pVal.Cotan_tot$value <- as.numeric(onlyPositive.pVal.Cotan_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultCOTAN <- roc(onlyPositive.pVal.Cotan_tot$value, 1 - onlyPositive.pVal.Cotan_tot$p_values)

# Plot the ROC curve
#plot(roc_resultCOTAN)

ROC for Seurat

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

# Plot the ROC curve
#plot(roc_resultSeurat)

ROC for Seurat scTransform

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_ScTransform_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat_scTr <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

ROC for Seurat Bimod

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_Bimod_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat_Bimod <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

ROC for Monocle

deaMonocle_tot <- NA
for (ind in 1:dim(subset.datasets_csv)[1]) {
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))
  
  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  #print(file.code)
  
  deaMonocle <- read.csv(file.path(dirOut,paste0(file.code,"Monocle_DEA_genes.csv")),row.names = 1) 
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaMonocle[deaMonocle$cell_group == cl.val,"cell_group"] <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaMonocle) <- str_replace(colnames(deaMonocle),
                                     pattern = "cell_group",
                                     replacement = "clusters") 
  colnames(deaMonocle) <- str_replace(colnames(deaMonocle),
                                     pattern = "gene_id",
                                     replacement = "genes")
  
  deaMonocle$data_set <- subset.datasets_csv[ind,1]
  deaMonocle$set_number <- ind 
  deaMonocle <- as.data.frame(deaMonocle)
  deaMonocle_tot <- rbind(deaMonocle_tot, deaMonocle)
  
  
  }
 deaMonocle_tot <-  deaMonocle_tot[2:nrow(deaMonocle_tot),]
 
 deaMonocle_tot <- merge.data.frame(deaMonocle_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaMonocle_tot$value <- as.numeric(deaMonocle_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultMonocle <- roc(deaMonocle_tot$value, 1 - deaMonocle_tot$marker_test_p_value)

# Plot the ROC curve
#plot(roc_resultMonocle)

ROC from Scanpy

deaScanpy_tot <- NA
for (ind in 1:dim(subset.datasets_csv)[1]) {
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  #print(file.code)
  
  deaScanpy <- read.csv(file.path(dirOut,paste0(file.code,"Scanpy_DEA_genes.csv")),
                        row.names = 1) 
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaScanpy[deaScanpy$clusters == paste0("cl",cl.val),"clusters"] <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)


  deaScanpy$data_set <- subset.datasets_csv[ind,1]
  deaScanpy$set_number <- ind 
  deaScanpy_tot <- rbind(deaScanpy_tot, deaScanpy)
  
  
  }
 deaScanpy_tot <-  deaScanpy_tot[2:nrow(deaScanpy_tot),]
 
 deaScanpy_tot <- merge.data.frame(deaScanpy_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaScanpy_tot$value <- as.numeric(deaScanpy_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultScanpy <- roc(deaScanpy_tot$value, 1 - deaScanpy_tot$pval)

# Plot the ROC curve
#plot(roc_resultScanpy)

Summary ROC for all methods

Three_Clusters_even <- ggroc(list(COTAN=roc_resultCOTAN, 
                                    Seurat=roc_resultSeurat,
                 Seurat_scTr = roc_resultSeurat_scTr, 
                 Seurat_Bimod = roc_resultSeurat_Bimod,
                 Monocle=roc_resultMonocle, 
                 Scanpy=roc_resultScanpy),
                 aes = c("colour"),
            legacy.axes = T,
            linewidth = 1)+theme_light()+
  scale_colour_manual("", 
                      values = methods.color)

Three_Clusters_evenPL <- Three_Clusters_even + 
  xlab("FPR") + ylab("TPR")+theme(legend.position="none")
Three_Clusters_even

3_Clusters_uneven

True vector

subset.datasets_csv <-datasets_csv[datasets_csv$Group == "3_Clusters_uneven",] 

ground_truth_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  clusters <- str_split(subset.datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  file.presence.subset <- file.presence[,clusters]
  
  
  #file.presence.subset <- as.matrix(file.presence.subset)
  
  
  ground_truth <- as.data.frame(matrix(nrow = nrow(file.presence.subset),
                                       ncol = ncol(file.presence.subset)))
  rownames(ground_truth) <- rownames(file.presence.subset)
  colnames(ground_truth) <- colnames(file.presence.subset)
  
  ground_truth[file.presence.subset == "Absent"] <- 0
  ground_truth[file.presence.subset == "Present"] <- 1
  ground_truth[file.presence.subset == "Uncertain"] <- 0.6
  
  file.presence.subset <- ground_truth
  
  for (col in 1:ncol(ground_truth)) {
    ground_truth[,col] <- FALSE  
    ground_truth[file.presence.subset[,col] == 1 & rowMeans(file.presence.subset[,-col,drop = FALSE]) < 0.35  ,col] <- TRUE
    }
  
  ground_truth$genes <- rownames(ground_truth) 
  ground_truth <- pivot_longer(ground_truth,
                               cols = 1:(ncol(ground_truth)-1),
                               names_to = "clusters")
  ground_truth$data_set <- subset.datasets_csv[ind,1]
  ground_truth$set_number <- ind 
  ground_truth_tot <- rbind(ground_truth_tot, ground_truth)
  
}
ground_truth_tot <- ground_truth_tot[2:nrow(ground_truth_tot),]

head(ground_truth_tot)
# A tibble: 6 × 5
  genes clusters  value data_set          set_number
  <chr> <chr>     <lgl> <chr>                  <int>
1 Neil2 E15.0-428 FALSE 3_Clusters_uneven          1
2 Neil2 E13.5-434 FALSE 3_Clusters_uneven          1
3 Neil2 E15.0-510 FALSE 3_Clusters_uneven          1
4 Lamc1 E15.0-428 FALSE 3_Clusters_uneven          1
5 Lamc1 E13.5-434 FALSE 3_Clusters_uneven          1
6 Lamc1 E15.0-510 FALSE 3_Clusters_uneven          1
length(unique(ground_truth_tot$genes)) 
[1] 14695
sum(ground_truth_tot$value)
[1] 1804

ROC for COTAN

onlyPositive.pVal.Cotan_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",
                      subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))
  deaCOTAN <- getClusterizationData(dataset,clName = "mergedClusters")[[2]]
  pvalCOTAN <- pValueFromDEA(deaCOTAN,
              numCells = getNumCells(dataset),method = "none")
  
  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.names <- c(cl.names,
                  str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1])
    cl.names <- cl.names[!is.na(cl.names)]
  }
  colnames(deaCOTAN) <- cl.names
  colnames(pvalCOTAN) <- cl.names
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  onlyPositive.pVal.Cotan <- pvalCOTAN

    for (cl in cl.names) {
    print(cl)
    #temp.DEA.CotanSign <- deaCOTAN[rownames(pvalCOTAN[pvalCOTAN[,cl] < 0.05,]) ,]
    onlyPositive.pVal.Cotan[rownames(deaCOTAN[deaCOTAN[,cl] < 0,]),cl] <-  1 #onlyPositive.pVal.Cotan[rownames(deaCOTAN[deaCOTAN[,cl] < 0,]),cl]+1
    }
    
    onlyPositive.pVal.Cotan$genes <- rownames(onlyPositive.pVal.Cotan) 
    onlyPositive.pVal.Cotan <- pivot_longer(onlyPositive.pVal.Cotan,
                                            values_to = "p_values",
                               cols = 1:(ncol(onlyPositive.pVal.Cotan)-1),
                               names_to = "clusters")
  onlyPositive.pVal.Cotan$data_set <- subset.datasets_csv[ind,1]
  onlyPositive.pVal.Cotan$set_number <- ind 
  onlyPositive.pVal.Cotan_tot <- rbind(onlyPositive.pVal.Cotan_tot, onlyPositive.pVal.Cotan)
  
  }
[1] "E15.0-510"
[1] "E13.5-434"
[1] "E15.0-428"
[1] "E15.0-432"
[1] "E13.5-432"
[1] "E13.5-187"
[1] "E15.0-509"
[1] "E15.0-508"
[1] "E13.5-184"
onlyPositive.pVal.Cotan_tot <- onlyPositive.pVal.Cotan_tot[2:nrow(onlyPositive.pVal.Cotan_tot),]

onlyPositive.pVal.Cotan_tot <- merge.data.frame(onlyPositive.pVal.Cotan_tot,
                                                ground_truth_tot,by = c("genes","clusters","data_set","set_number"),all.x = T,all.y = F)

#onlyPositive.pVal.Cotan_tot <- onlyPositive.pVal.Cotan_tot[order(onlyPositive.pVal.Cotan_tot$p_values,
 #                                                                decreasing = F),]
# df <- as.data.frame(matrix(nrow = nrow(onlyPositive.pVal.Cotan_tot),ncol = 3)) 
# colnames(df) <- c("TPR","FPR","Method")
# df$Method <- "COTAN"
# 
# Positive <- sum(onlyPositive.pVal.Cotan_tot$value) 
# Negative <- sum(!onlyPositive.pVal.Cotan_tot$value)
# 
# for (i in 1:nrow(onlyPositive.pVal.Cotan_tot)) {
#   df[i,"TPR"] <- sum(onlyPositive.pVal.Cotan_tot[1:i,"value"])/Positive
#   df[i,"FPR"] <- (i-sum(onlyPositive.pVal.Cotan_tot[1:i,"value"]))/Negative
  
# Convert TRUE/FALSE to 1/0
onlyPositive.pVal.Cotan_tot$value <- as.numeric(onlyPositive.pVal.Cotan_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultCOTAN <- roc(onlyPositive.pVal.Cotan_tot$value, 1 - onlyPositive.pVal.Cotan_tot$p_values)

# Plot the ROC curve
#plot(roc_resultCOTAN)

ROC for Seurat

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

# Plot the ROC curve
#plot(roc_resultSeurat)

ROC for Seurat scTransform

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_ScTransform_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat_scTr <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

ROC for Seurat Bimod

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_Bimod_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat_Bimod <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

ROC for Monocle

deaMonocle_tot <- NA
for (ind in 1:dim(subset.datasets_csv)[1]) {
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))
  
  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  #print(file.code)
  
  deaMonocle <- read.csv(file.path(dirOut,paste0(file.code,"Monocle_DEA_genes.csv")),row.names = 1) 
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaMonocle[deaMonocle$cell_group == cl.val,"cell_group"] <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaMonocle) <- str_replace(colnames(deaMonocle),
                                     pattern = "cell_group",
                                     replacement = "clusters") 
  colnames(deaMonocle) <- str_replace(colnames(deaMonocle),
                                     pattern = "gene_id",
                                     replacement = "genes")
  
  deaMonocle$data_set <- subset.datasets_csv[ind,1]
  deaMonocle$set_number <- ind 
  deaMonocle <- as.data.frame(deaMonocle)
  deaMonocle_tot <- rbind(deaMonocle_tot, deaMonocle)
  
  
  }
 deaMonocle_tot <-  deaMonocle_tot[2:nrow(deaMonocle_tot),]
 
 deaMonocle_tot <- merge.data.frame(deaMonocle_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaMonocle_tot$value <- as.numeric(deaMonocle_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultMonocle <- roc(deaMonocle_tot$value, 1 - deaMonocle_tot$marker_test_p_value)

# Plot the ROC curve
#plot(roc_resultMonocle)

ROC from Scanpy

deaScanpy_tot <- NA
for (ind in 1:dim(subset.datasets_csv)[1]) {
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  #print(file.code)
  
  deaScanpy <- read.csv(file.path(dirOut,paste0(file.code,"Scanpy_DEA_genes.csv")),
                        row.names = 1) 
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaScanpy[deaScanpy$clusters == paste0("cl",cl.val),"clusters"] <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)


  deaScanpy$data_set <- subset.datasets_csv[ind,1]
  deaScanpy$set_number <- ind 
  deaScanpy_tot <- rbind(deaScanpy_tot, deaScanpy)
  
  
  }
 deaScanpy_tot <-  deaScanpy_tot[2:nrow(deaScanpy_tot),]
 
 deaScanpy_tot <- merge.data.frame(deaScanpy_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaScanpy_tot$value <- as.numeric(deaScanpy_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultScanpy <- roc(deaScanpy_tot$value, 1 - deaScanpy_tot$pval)

# Plot the ROC curve
#plot(roc_resultScanpy)

Summary ROC for all methods

Three_Clusters_uneven <- ggroc(list(COTAN=roc_resultCOTAN, 
                                    Seurat=roc_resultSeurat,
                 Seurat_scTr = roc_resultSeurat_scTr, 
                 Seurat_Bimod = roc_resultSeurat_Bimod,
                 Monocle=roc_resultMonocle, 
                 Scanpy=roc_resultScanpy),
                 aes = c("colour"),
            legacy.axes = T,
            linewidth = 1)+theme_light()+
  scale_colour_manual("", 
                      values = methods.color)

Three_Clusters_unevenPL <- Three_Clusters_uneven + xlab("FPR") + ylab("TPR")+theme(legend.position="none")

Three_Clusters_uneven

5 clusters

5_Clusters_uneven

True vector

subset.datasets_csv <-datasets_csv[datasets_csv$Group == "5_Clusters_uneven",] 

ground_truth_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  clusters <- str_split(subset.datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  file.presence.subset <- file.presence[,clusters]
  
  
  #file.presence.subset <- as.matrix(file.presence.subset)
  
  
  ground_truth <- as.data.frame(matrix(nrow = nrow(file.presence.subset),
                                       ncol = ncol(file.presence.subset)))
  rownames(ground_truth) <- rownames(file.presence.subset)
  colnames(ground_truth) <- colnames(file.presence.subset)
  
  ground_truth[file.presence.subset == "Absent"] <- 0
  ground_truth[file.presence.subset == "Present"] <- 1
  ground_truth[file.presence.subset == "Uncertain"] <- 0.6
  
  file.presence.subset <- ground_truth
  
  for (col in 1:ncol(ground_truth)) {
    ground_truth[,col] <- FALSE  
    ground_truth[file.presence.subset[,col] == 1 & rowMeans(file.presence.subset[,-col,drop = FALSE]) < 0.35  ,col] <- TRUE
    }
  
  ground_truth$genes <- rownames(ground_truth) 
  ground_truth <- pivot_longer(ground_truth,
                               cols = 1:(ncol(ground_truth)-1),
                               names_to = "clusters")
  ground_truth$data_set <- subset.datasets_csv[ind,1]
  ground_truth$set_number <- ind 
  ground_truth_tot <- rbind(ground_truth_tot, ground_truth)
  
}
ground_truth_tot <- ground_truth_tot[2:nrow(ground_truth_tot),]



head(ground_truth_tot)
# A tibble: 6 × 5
  genes clusters  value data_set          set_number
  <chr> <chr>     <lgl> <chr>                  <int>
1 Neil2 E13.5-510 FALSE 5_Clusters_uneven          1
2 Neil2 E15.0-437 FALSE 5_Clusters_uneven          1
3 Neil2 E15.0-510 FALSE 5_Clusters_uneven          1
4 Neil2 E13.5-432 FALSE 5_Clusters_uneven          1
5 Neil2 E13.5-437 FALSE 5_Clusters_uneven          1
6 Lamc1 E13.5-510 FALSE 5_Clusters_uneven          1
length(unique(ground_truth_tot$genes)) 
[1] 14695
sum(ground_truth_tot$value)
[1] 1663

ROC for COTAN

onlyPositive.pVal.Cotan_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",
                      subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))
  deaCOTAN <- getClusterizationData(dataset,clName = "mergedClusters")[[2]]
  pvalCOTAN <- pValueFromDEA(deaCOTAN,
              numCells = getNumCells(dataset),method = "none")
  genesLambda <- getLambda(dataset)
  genesLambda <- as.data.frame(genesLambda)
  genesLambda$genes <- rownames(genesLambda)
  
  
  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.names <- c(cl.names,
                  str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1])
    cl.names <- cl.names[!is.na(cl.names)]
  }
  colnames(deaCOTAN) <- cl.names
  colnames(pvalCOTAN) <- cl.names
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  onlyPositive.pVal.Cotan <- pvalCOTAN

    for (cl in cl.names) {
    print(cl)
    #temp.DEA.CotanSign <- deaCOTAN[rownames(pvalCOTAN[pvalCOTAN[,cl] < 0.05,]) ,]
    onlyPositive.pVal.Cotan[rownames(deaCOTAN[deaCOTAN[,cl] < 0,]),cl] <-  1 #onlyPositive.pVal.Cotan[rownames(deaCOTAN[deaCOTAN[,cl] < 0,]),cl]+1
    }
    
    onlyPositive.pVal.Cotan$genes <- rownames(onlyPositive.pVal.Cotan) 
    onlyPositive.pVal.Cotan <- pivot_longer(onlyPositive.pVal.Cotan,
                                            values_to = "p_values",
                               cols = 1:(ncol(onlyPositive.pVal.Cotan)-1),
                               names_to = "clusters")
  onlyPositive.pVal.Cotan$data_set <- subset.datasets_csv[ind,1]
  onlyPositive.pVal.Cotan$set_number <- ind 
  
  onlyPositive.pVal.Cotan <- merge(onlyPositive.pVal.Cotan,genesLambda,by="genes")
  
  onlyPositive.pVal.Cotan_tot <- rbind(onlyPositive.pVal.Cotan_tot, onlyPositive.pVal.Cotan)
  
  }
[1] "E13.5-432"
[1] "E15.0-510"
[1] "E13.5-437"
[1] "E15.0-437"
[1] "E13.5-510"
[1] "E13.5-434"
[1] "E15.0-428"
[1] "E13.5-184"
[1] "E15.0-434"
[1] "E17.5-505"
[1] "E15.0-432"
[1] "E13.5-432"
[1] "E15.0-509"
[1] "E15.0-508"
[1] "E13.5-187"
onlyPositive.pVal.Cotan_tot <- onlyPositive.pVal.Cotan_tot[2:nrow(onlyPositive.pVal.Cotan_tot),]

onlyPositive.pVal.Cotan_tot <- merge.data.frame(onlyPositive.pVal.Cotan_tot,
                                                ground_truth_tot,by = c("genes","clusters","data_set","set_number"),all.x = T,all.y = F)


  
# Convert TRUE/FALSE to 1/0
onlyPositive.pVal.Cotan_tot$value <- as.numeric(onlyPositive.pVal.Cotan_tot$value)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultCOTAN <- roc(onlyPositive.pVal.Cotan_tot$value, 1 - onlyPositive.pVal.Cotan_tot$p_values)

cotan.threshold.pval <- quantile(onlyPositive.pVal.Cotan_tot[onlyPositive.pVal.Cotan_tot$value == 0,]$p_values,probs = 0.05)

# Plot the ROC curve
#plot(roc_resultCOTAN)

ROC for Seurat

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

deaSeurat_tot_base <- deaSeurat_tot

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)


seurat.threshold.pval <- quantile(deaSeurat_tot_base[deaSeurat_tot_base$value == 0,]$p_val,probs = 0.05)
# Plot the ROC curve
#plot(roc_resultSeurat)

ROC for Seurat scTransform

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_ScTransform_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

deaSeurat_tot_scTr <- deaSeurat_tot

Seurat_scTr.threshold.pval <- quantile(deaSeurat_tot_scTr[deaSeurat_tot_scTr$value == 0,]$p_val,probs = 0.05)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat_scTr <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

ROC for Seurat Bimod

deaSeurat_tot <- NA

for (ind in 1:dim(subset.datasets_csv)[1]) {
  #print(ind)
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  
  deaSeurat <- read.csv(file.path(dirOut,paste0(file.code,"Seurat_DEA_Bimod_genes.csv")), row.names = 1)
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaSeurat[deaSeurat$cluster == cl.val,]$cluster <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "cluster",
                                     replacement = "clusters") 
  colnames(deaSeurat) <- str_replace(colnames(deaSeurat),
                                     pattern = "gene",
                                     replacement = "genes") 
  
  deaSeurat$data_set <- subset.datasets_csv[ind,1]
  deaSeurat$set_number <- ind 
  deaSeurat_tot <- rbind(deaSeurat_tot, deaSeurat)
  
  
  }
 deaSeurat_tot <-  deaSeurat_tot[2:nrow(deaSeurat_tot),]
 
 deaSeurat_tot <- merge.data.frame(deaSeurat_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaSeurat_tot$value <- as.numeric(deaSeurat_tot$value)

deaSeurat_tot_Bimod <- deaSeurat_tot

seurat_bimod.threshold.pval <- quantile(deaSeurat_tot_Bimod[deaSeurat_tot_Bimod$value == 0,]$p_val,probs = 0.05)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultSeurat_Bimod <- roc(deaSeurat_tot$value, 1 - deaSeurat_tot$p_val)

ROC for Monocle

deaMonocle_tot <- NA
for (ind in 1:dim(subset.datasets_csv)[1]) {
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))
  
  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  #print(file.code)
  
  deaMonocle <- read.csv(file.path(dirOut,paste0(file.code,"Monocle_DEA_genes.csv")),row.names = 1) 
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaMonocle[deaMonocle$cell_group == cl.val,"cell_group"] <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)
  
  colnames(deaMonocle) <- str_replace(colnames(deaMonocle),
                                     pattern = "cell_group",
                                     replacement = "clusters") 
  colnames(deaMonocle) <- str_replace(colnames(deaMonocle),
                                     pattern = "gene_id",
                                     replacement = "genes")
  
  deaMonocle$data_set <- subset.datasets_csv[ind,1]
  deaMonocle$set_number <- ind 
  deaMonocle <- as.data.frame(deaMonocle)
  deaMonocle_tot <- rbind(deaMonocle_tot, deaMonocle)
  
  
  }
 deaMonocle_tot <-  deaMonocle_tot[2:nrow(deaMonocle_tot),]
 
 deaMonocle_tot <- merge.data.frame(deaMonocle_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaMonocle_tot$value <- as.numeric(deaMonocle_tot$value)

monocle.threshold.pval <- quantile(deaMonocle_tot[deaMonocle_tot$value == 0,]$marker_test_p_value,probs = 0.05)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultMonocle <- roc(deaMonocle_tot$value, 1 - deaMonocle_tot$marker_test_p_value)

# Plot the ROC curve
#plot(roc_resultMonocle)

ROC from Scanpy

deaScanpy_tot <- NA
for (ind in 1:dim(subset.datasets_csv)[1]) {
  file.code <- paste0(subset.datasets_csv$Group[ind],"_",subset.datasets_csv$Collection[ind])
  dataset <- readRDS(file = file.path(dataSetDir,paste0(file.code,".RDS")))

  clusterization <- getClusterizationData(dataset, clName = "mergedClusters")[[1]]
  #print(file.code)
  
  deaScanpy <- read.csv(file.path(dirOut,paste0(file.code,"Scanpy_DEA_genes.csv")),
                        row.names = 1) 
  
  cl.names <- NA
  
  for (cl.val in  unique(clusterization)) {
    #print(cl.val)
    cl.name <- str_split(names(clusterization[clusterization == cl.val])[1],
                            pattern = "_",simplify = T)[1]
    cl.names <- c(cl.names,cl.name)
    cl.names <- cl.names[!is.na(cl.names)]
  
    deaScanpy[deaScanpy$clusters == paste0("cl",cl.val),"clusters"] <- cl.name   
  
  }
  
  clusters <- str_split(datasets_csv$Collection[ind],pattern = "_[+]_",simplify = T)


  deaScanpy$data_set <- subset.datasets_csv[ind,1]
  deaScanpy$set_number <- ind 
  deaScanpy_tot <- rbind(deaScanpy_tot, deaScanpy)
  
  
  }
 deaScanpy_tot <-  deaScanpy_tot[2:nrow(deaScanpy_tot),]
 
 deaScanpy_tot <- merge.data.frame(deaScanpy_tot,
                                   ground_truth_tot,
                                   by = c("genes","clusters","data_set","set_number"),
                                   all.x = T,all.y = F)

# Convert TRUE/FALSE to 1/0
deaScanpy_tot$value <- as.numeric(deaScanpy_tot$value)

scanpy.threshold.pval <- quantile(deaScanpy_tot[deaScanpy_tot$value == 0,]$pval,probs = 0.05)

# Compute the ROC curve - note that we invert the p-values with 1 - p_values
roc_resultScanpy <- roc(deaScanpy_tot$value, 1 - deaScanpy_tot$pval)

# Plot the ROC curve
#plot(roc_resultScanpy)

Summary ROC for all methods

Five_Clusters <- ggroc(list(COTAN=roc_resultCOTAN, 
                                    Seurat=roc_resultSeurat,
                 Seurat_scTr = roc_resultSeurat_scTr, 
                 Seurat_Bimod = roc_resultSeurat_Bimod,
                 Monocle=roc_resultMonocle, 
                 Scanpy=roc_resultScanpy),
                 aes = c("colour"),
            legacy.axes = T,
            linewidth = 1)+theme_light()+
  scale_colour_manual("", 
                      values = methods.color)

 
Five_Clusters_unevenPL <- Five_Clusters + xlab("FPR") + ylab("TPR")

Five_Clusters_unevenPL

Global Summary

2 Clusters

plot_2cl <- ggarrange(TwoClusters_even_nearPL,Two_Clusters_even_mediumPL, Two_Clusters_even_farPL,Two_Clusters_uneven_nearPL, Two_Clusters_uneven_mediumPL,Two_Clusters_uneven_farPL,
          labels = c("Even_Near", "Even_Medium", "Even_Far", "Uneven_Near","Uneven_Medium","Uneven_Far"),
          ncol = 3, nrow = 2, common.legend = T, legend = "bottom")

pdf(paste0(dirOut,"ROC_two_supp.pdf"),width = 15,height = 10)
plot(plot_2cl)
dev.off()
png 
  2 
plot_2cl

3 and 5 Clusters

plot_3_5 <- ggarrange(Three_Clusters_evenPL,Three_Clusters_unevenPL, NULL, Five_Clusters_unevenPL,
          labels = c("3_Even", "3_Uneven", "", "5_Uneven"),
          ncol = 2, nrow = 2, common.legend = T, legend = "bottom")

pdf(paste0(dirOut,"ROC_three_five_supp.pdf"),width = 10,height = 10)
plot(plot_3_5)
dev.off()
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plot_3_5

plot3 <- ggarrange(Two_Clusters_uneven_farPL,Three_Clusters_unevenPL, Five_Clusters_unevenPL,
          labels = c("A", "B", "C"),
          ncol = 3, nrow = 1, common.legend = T, legend = "none")

pdf(paste0(dirOut,"ROC_three_main.pdf"),width = 15,height = 5)
plot(plot3)
dev.off()
png 
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plot3

Histogram with gene expression level

ground_truth_tot_subset <- as.data.frame(ground_truth_tot[ground_truth_tot$value ==1,])

COTAN_data <- onlyPositive.pVal.Cotan_tot[,c("genes","set_number","clusters","p_values","genesLambda")]

colnames(COTAN_data) <- c("genes","set_number","clusters","p_value_COTAN","genesLambda")

ground_truth_tot_subset <- merge(ground_truth_tot_subset,
                                 COTAN_data,
                                 by=c("genes","set_number", "clusters"),
                                 all.x = T, all.y= FALSE)

Seurat_data_base <- deaSeurat_tot_base[,c("genes","set_number","clusters", "p_val")]
colnames(Seurat_data_base) <- c("genes","set_number","clusters","p_value_Seurat")

ground_truth_tot_subset <- merge(ground_truth_tot_subset,
                                 Seurat_data_base,
                                 by=c("genes","set_number","clusters"),
                                 all.x = T, all.y= FALSE)

Seurat_data_scTr <- deaSeurat_tot_scTr[,c("genes","set_number","clusters", "p_val")]
colnames(Seurat_data_scTr) <- c("genes","set_number","clusters","p_value_Seurat_scTr")

ground_truth_tot_subset <- merge(ground_truth_tot_subset,
                                 Seurat_data_scTr,
                                 by=c("genes","set_number","clusters"),
                                 all.x = T, all.y= FALSE)

Seurat_data_Bimod <- deaSeurat_tot_Bimod[,c("genes","set_number","clusters", "p_val")]
colnames(Seurat_data_Bimod) <- c("genes","set_number","clusters","p_value_Seurat_Bimod")

ground_truth_tot_subset <- merge(ground_truth_tot_subset,
                                 Seurat_data_Bimod,
                                 by=c("genes","set_number","clusters"),
                                 all.x = T, all.y= FALSE)


data_Monocle <- deaMonocle_tot[,c("genes","set_number","clusters", "marker_test_p_value")]
colnames(data_Monocle) <- c("genes","set_number","clusters","p_value_Monocle")

ground_truth_tot_subset <- merge(ground_truth_tot_subset,
                                 data_Monocle,
                                 by=c("genes","set_number","clusters"),
                                 all.x = T, all.y= FALSE)

data_Scanpy <- deaScanpy_tot[,c("genes","set_number","clusters", "pval")]
colnames(data_Scanpy) <- c("genes","set_number","clusters","p_value_Scanpy")

ground_truth_tot_subset <- merge(ground_truth_tot_subset,
                                 data_Scanpy,
                                 by=c("genes","set_number","clusters"),
                                 all.x = T, all.y= FALSE)

ground_truth_tot_subset <- ground_truth_tot_subset %>% mutate(lambda_bin = cut(genesLambda, breaks=50))

ground_truth_tot_subset[is.na(ground_truth_tot_subset)] <- 1

head(ground_truth_tot_subset)
          genes set_number  clusters value          data_set p_value_COTAN
1 A330102I10Rik          2 E17.5-505  TRUE 5_Clusters_uneven  3.580400e-01
2 A630089N07Rik          2 E13.5-184  TRUE 5_Clusters_uneven  1.152963e-10
3 A830010M20Rik          1 E15.0-510  TRUE 5_Clusters_uneven  6.731088e-05
4 A930004D18Rik          2 E13.5-184  TRUE 5_Clusters_uneven  1.847177e-04
5         Aaed1          3 E13.5-187  TRUE 5_Clusters_uneven  1.613229e-05
6          Abat          2 E15.0-428  TRUE 5_Clusters_uneven  4.997729e-07
  genesLambda p_value_Seurat p_value_Seurat_scTr p_value_Seurat_Bimod
1  0.03431373   1.000000e+00        1.000000e+00         2.718294e-04
2  0.06617647   1.632048e-12        7.812652e-13         1.731541e-09
3  0.07175926   2.117000e-07        1.205805e-07         1.913975e-06
4  0.06740196   1.841516e-05        1.287232e-05         4.368518e-06
5  0.01704158   8.730213e-07        7.218352e-07         3.744192e-05
6  0.05514706   2.469818e-05        3.984286e-05         5.906623e-04
  p_value_Monocle p_value_Scanpy       lambda_bin
1    1.935776e-01    0.793509146  (0.0341,0.0641]
2    1.328598e-09    0.004754579  (0.0641,0.0941]
3    2.189873e-06    0.024391612  (0.0641,0.0941]
4    1.947179e-04    0.074967388  (0.0641,0.0941]
5    2.742404e-03    0.311500465 (0.00259,0.0341]
6    6.040666e-05    0.102450927  (0.0341,0.0641]
bins <- levels(ground_truth_tot_subset$lambda_bin)
bins <- bins[!is.na(bins)]

forBarChart <- as.data.frame(matrix(nrow = length(bins),ncol = 7,data = 0))

colnames(forBarChart) <- c("bins","COTAN","Seurat","Seurat_Bimod","Seurat_scTr","Monocle", "Scanpy")
forBarChart$bins <- bins
rownames(forBarChart) <- forBarChart$bins

#p_val_threshold <- 0.05

for (i in as.vector(forBarChart$bins)) {
  forBarChart[ i,"COTAN"] <- sum(ground_truth_tot_subset$lambda_bin == i & ground_truth_tot_subset$p_value_COTAN <= cotan.threshold.pval)
  
  forBarChart[ i,"Seurat"] <- sum(ground_truth_tot_subset$lambda_bin == i & ground_truth_tot_subset$p_value_Seurat <= seurat.threshold.pval)
  
  forBarChart[ i,"Seurat_Bimod"] <- sum(ground_truth_tot_subset$lambda_bin == i & ground_truth_tot_subset$p_value_Seurat_Bimod <= seurat_bimod.threshold.pval)
  
  forBarChart[ i,"Seurat_scTr"] <- sum(ground_truth_tot_subset$lambda_bin == i & ground_truth_tot_subset$p_value_Seurat_scTr <= Seurat_scTr.threshold.pval)
  
  forBarChart[ i,"Monocle"] <- sum(ground_truth_tot_subset$lambda_bin == i & ground_truth_tot_subset$p_value_Monocle <= monocle.threshold.pval)
  
  forBarChart[ i,"Scanpy"] <- sum(ground_truth_tot_subset$lambda_bin == i & ground_truth_tot_subset$p_value_Scanpy <= scanpy.threshold.pval)
  
}

temp.bin <- as.data.frame(str_split(forBarChart$bins,pattern = ",",simplify = T))
temp.bin[,1] <- as.numeric(str_remove(temp.bin[,1],pattern = "[(]"))
temp.bin[,2] <- as.numeric(str_remove(temp.bin[,2],pattern = "[]]"))

forBarChart$bins <- round(rowMeans(temp.bin),digits = 2)


forBarChart <- forBarChart[rowSums(forBarChart[,2:ncol(forBarChart)]) > 0,]

forBarChart.perc <- forBarChart[1:8,]

forBarChart.perc$tot <- rowSums(forBarChart.perc[,2:ncol(forBarChart.perc)])

forBarChart.perc[,2:(ncol(forBarChart.perc)-1)] <- round(forBarChart.perc[,2:(ncol(forBarChart.perc)-1)]/forBarChart.perc$tot,digits = 4)

forBarChart.perc <- pivot_longer(forBarChart.perc,
                               cols = 2:(ncol(forBarChart.perc)-1),
                               names_to = "Methods")

forBarChart.number <- pivot_longer(forBarChart[1:8,],
                               cols = 2:(ncol(forBarChart)),
                               names_to = "Methods")

colnames(forBarChart.number) <- c("bins","Methods", "valueOriginal")

forBarChart.perc <- merge(forBarChart.perc,forBarChart.number, by = c("bins","Methods"))

barChart <- ggplot(forBarChart.perc,aes(x=bins,y=value,fill=Methods))+
  geom_bar(stat="identity")+
  geom_text(aes(label = valueOriginal), 
            position = position_stack(vjust = 0.5),  size=3)+
  scale_fill_manual(
    values = methods.color)+
  theme_light()+
  scale_x_continuous(breaks =forBarChart.perc$bins)+
  theme(axis.title.y=element_blank(),axis.title.x=element_blank(),
      legend.position="none",axis.text.x = element_text(angle = 45, hjust=1)
    )+
  scale_y_continuous(labels = scales::percent_format())

pdf(paste0(dirOut,"EnrichedGenesForExpressionLevel.pdf"),height = 3,width = 5)
barChart
dev.off()
png 
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plot(barChart)

plot.with.inset <-
  ggdraw() +
  draw_plot(plot3) +
  draw_plot(as_ggplot(lg), x = 0.18, y = 0.1, width = 0.2, height = .45)+
  draw_plot(barChart, x = 0.78, y = 0.09, width = 0.22, height = .5)

pdf(paste0(dirOut,"Plot3WihtGenesExLevel.pdf"),height = 5,width = 15)
plot(plot.with.inset)
dev.off()
png 
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plot(plot.with.inset)