sc-TissueMapper_functions.R 34.8 KB
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#sc-TissueMapper
#Author: Gervaise H. Henry
#Email: gervaise.henry@utsouthwestern.edu
#Lab: Strand Lab, Deparment of Urology, University of Texas Southwestern Medical Center


scFolders <- function(){
  if (!dir.exists("./analysis/qc/")){
    dir.create("./analysis/qc/")
  }
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  if (!dir.exists("./analysis/qc/")){
    dir.create("./analysis/qc/")
  }
  if (!dir.exists("./analysis/qc/cutoffs/")){
    dir.create("./analysis/qc/cutoffs/")
  }
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  if (!dir.exists("./analysis/qc/cellcycle")){
    dir.create("./analysis/qc/cellcycle")
  }
  if (!dir.exists("./analysis/vis")){
    dir.create("./analysis/vis")
  }
  if (!dir.exists("./analysis/score_id")){
    dir.create("./analysis/score_id")
  }
  if (!dir.exists("./analysis/cor")){
    dir.create("./analysis/cor")
  }
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  if (!dir.exists("./analysis/shiny")){
    dir.create("./analysis/shiny")
  }
  if (!dir.exists("./analysis/shiny")){
    dir.create("./analysis/shiny")
  }
  if (!dir.exists("./analysis/shiny/raw")){
    dir.create("./analysis/shiny/raw")
  }
  if (!dir.exists("./analysis/shiny/raw/outs")){
    dir.create("./analysis/shiny/raw/outs")
  }
  if (!dir.exists("./analysis/shiny/raw/outs/analysis")){
    dir.create("./analysis/shiny/raw/outs/analysis")
  }
  if (!dir.exists("./analysis/shiny/raw/outs/analysis/clustering")){
    dir.create("./analysis/shiny/raw/outs/analysis/clustering")
  }
  if (!dir.exists("./analysis/shiny/raw/outs/analysis/diffexp")){
    dir.create("./analysis/shiny/raw/outs/analysis/diffexp")
  }
  if (!dir.exists("./analysis/shiny/raw/outs/analysis/pca")){
    dir.create("./analysis/shiny/raw/outs/analysis/pca")
  }
  if (!dir.exists("./analysis/shiny/raw/outs/analysis/pca/10_components")){
    dir.create("./analysis/shiny/raw/outs/analysis/pca/10_components")
  }
  if (!dir.exists("./analysis/shiny/raw/outs/analysis/tsne")){
    dir.create("./analysis/shiny/raw/outs/analysis/tsne")
  }
  if (!dir.exists("./analysis/shiny/raw/outs/analysis/tsne/2_components")){
    dir.create("./analysis/shiny/raw/outs/analysis/tsne/2_components")
  }
  if (!dir.exists("./analysis/shiny/raw/outs/analysis/umap")){
    dir.create("./analysis/shiny/raw/outs/analysis/umap")
  }
  if (!dir.exists("./analysis/shiny/raw/outs/analysis/umap/2_components")){
    dir.create("./analysis/shiny/raw/outs/analysis/umap/2_components")
  }
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}


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scLoad <- function(p,cellranger=3,aggr=TRUE){
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  #Load and prefilter filtered_gene_bc_matrices_mex output from cellranger
  
  #Inputs:
  #p = project name
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  #cellranger cellranger version number used for count/aggr, 2 or 3
  #aggr = if the samples are already aggregated, TRUE if useing the output of aggr, FALSE if using outputs of each count
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  #Outputs:
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  #sc10x = Seurat object list
  #sc10x.groups = group labels for each sample
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  sc10x.groups <- read_csv(paste0("./analysis/DATA/",p,"-demultiplex.csv"))
  
  
  #Load filtered_gene_bc_matrices output from cellranger
  sc10x.data <- list()
  sc10x <- list()
  if (aggr==TRUE){
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    if (cellranger==2){
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      sc10x.data[aggr] <- Read10X(data.dir=paste0("./analysis/DATA/10x/filtered_gene_bc_matrices_mex/"))
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    } else {
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      sc10x.data[aggr] <- Read10X(data.dir=paste0("./analysis/DATA/10x/filtered_feature_bc_matrix/"))
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    }
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    sc10x[aggr] <- new("seurat",raw.data=sc10x.data[aggr])
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  } else {
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    for (i in sc10x.groups$Samples){
      if (cellranger==2){
        sc10x.data[i] <- Read10X(data.dir=paste0("./analysis/DATA/10x/",i,"/filtered_gene_bc_matrices/"))
      } else {
        sc10x.data[i] <- Read10X(data.dir=paste0("./analysis/DATA/10x/",i,"/filtered_feature_bc_matrix/"))
      }
      sc10x[i] <- CreateSeuratObject(counts=sc10x.data[[i]],project=p)
      sc10x[[i]]$samples <- i
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    }
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  }
  
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  # #Label sample names from aggregation_csv.csv
  # if (sub==FALSE){
  #   if (cellranger==2){
  #     sc10x.aggr <- read_csv("./analysis/DATA/10x/aggregation_csv.csv")
  #   } else {
  #     sc10x.aggr <- read_csv("./analysis/DATA/10x/aggregation.csv")
  #   }
  # } else {
  #   if (cellranger==2){
  #     sc10x.aggr <- read_csv(paste0("./analysis/DATA/",p,"/10x/aggregation_csv.csv"))
  #   } else {
  #     sc10x.aggr <- read_csv(paste0("./analysis/DATA/",p,"/10x/aggregation.csv"))
  #   }
  # }
  # cell.codes <- as.data.frame(sc10x@raw.data@Dimnames[[2]])
  # colnames(cell.codes) <- "barcodes"
  # rownames(cell.codes) <- cell.codes$barcodes
  # cell.codes$lib.codes <- as.factor(gsub(pattern=".+-",replacement="",cell.codes$barcodes))
  # cell.codes$samples <- sc10x.aggr$library_id[match(cell.codes$lib.codes,as.numeric(rownames(sc10x.aggr)))]
  # sc10x <- CreateSeuratObject(counts=sc10x.data,project=p,assay="RNA",min.cells=mc,min.features=mg,meta.data=cell.codes["samples"])
  # 
  # #Create groups found in demultiplex.csv
  # for (i in 2:ncol(sc10x.demultiplex)){
  #   Idents(sc10x) <- "samples"
  #   merge.cluster <- apply(sc10x.demultiplex[,i],1,as.character)
  #   merge.cluster[merge.cluster==1] <- colnames(sc10x.demultiplex[,i])
  #   
  #   Idents(sc10x) <- plyr::mapvalues(x=Idents(sc10x),from=sc10x.demultiplex$Samples,to=merge.cluster)
  #   sc10x@meta.data[,colnames(sc10x.demultiplex[,i])] <- Idents(sc10x)
  # }
  
  
  results <- list(
    sc10x=sc10x,
    sc10x.groups=sc10x.groups
  )
  return(results)
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}


scSubset <- function(sc10x,i="ALL",g="ALL"){
  #Subset cells based on an identity
  
  #Inputs:
  #sc10x = seruat object
  #i = identity to use
  #g = group to subset by
  
  #Outputs:
  #Seurat object
  
  
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  Idents(sc10x) <- i
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  sc10x.sub <- subset(x=sc10x,idents=g)
  
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  return(sc10x.sub)
}


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scQC <- function(sc10x,sp="hu"){
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  #QC and filter Seurat object
  
  #Inputs:
  #sc10x = Seruat object
  #sub = Subfolder to save output files
  
  #Outputs:
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  #result[1] = filtered Seurat object
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  #result[2] = raw cell count
  #result[3] = raw gene count
  #result[4] = filtered cell count
  #result[5] = filtered gene count
  
  
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  #Calculate percent mitochondrea
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  if (sp=="hu"){
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    mito.pattern <- "^MT-"
  } else if (sp=="mu"){
    mito.pattern <- "^mt-"
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  }
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  for (i in names(sc10x)){
    sc10x.temp <- sc10x[[i]]
    sc10x.temp[["percent.mito"]] <- PercentageFeatureSet(object=sc10x.temp,pattern=mito.pattern)
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    #sc10x.temp <- subset(sc10x.temp,cell=names(which(is.na(sc10x.temp$percent.mito))),invert=TRUE)
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    sc10x[i] <- sc10x.temp
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  }
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  #Calculate cutoffs
  thresh <- list()
  h <- list()
  cells.remove <- list()
  sc10x.temp <- list()
  thresh.l <- list()
  cutoff.l.mito <- list()
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  for (i in c("nFeature_RNA","percent.mito")){
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    thresh[[i]] <- scThresh(sc10x,feature=i)
    if (i == "percent.mito"){
      for (j in names(sc10x)){
        cutoff.l.mito[[j]] <- NULL
        h[[i]] <- hist(data.frame(sc10x[[j]][[i]])$percent.mito,breaks=1000,plot=FALSE)
        cutoff.temp <- mean(c(h[[i]]$mids[which.max(h[[i]]$counts)],h[[i]]$mids[-which.max(h[[i]]$counts)][which.max(h[[i]]$counts[-which.max(h[[i]]$counts)])]))
        cells.remove[[j]] <- c(cells.remove[[j]],rownames(sc10x[[j]][["percent.mito"]])[sc10x[[j]][[i]][,1] > cutoff.temp])
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        sc10x.temp[[j]] <- subset(sc10x[[j]],cells=setdiff(colnames(sc10x[[j]]),cells.remove[[j]]))
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        thresh.l[[i]] <- scThresh(sc10x.temp,feature=i,sub="lower")
        cutoff.l.mito[[j]] <- thresh.l[[i]][[j]]$threshold[thresh.l[[i]][[j]]$method=="Triangle"]
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      }
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    }
  }
  
  #Plot raw stats
  max.ct <- 0
  max.ft <- 0
  max.mt <- 0
  for (i in names(sc10x)){
    if (max.ct < max(sc10x[[i]][["nCount_RNA"]])){
      max.ct <- max(sc10x[[i]][["nCount_RNA"]])
    }
    if (max.ft < max(sc10x[[i]][["nFeature_RNA"]])){
      max.ft <- max(sc10x[[i]][["nFeature_RNA"]])
    }
    if (max.mt < max(sc10x[[i]][["percent.mito"]])){
      max.mt <- max(sc10x[[i]][["percent.mito"]])
    }
  }
  max.ct <- max.ct*1.1
  max.ft <- max.ft*1.1
  max.mt <- max.mt*1.1
  cells.remove <- list()
  for (i in c("nCount_RNA","nFeature_RNA","percent.mito")){
    max.y <- 0
    if (i == "nCount_RNA"){
      cells.remove[[j]] <- NULL
      max.y <- max.ct
    } else if (i == "nFeature_RNA"){
      max.y <- max.ft
    } else if (i == "percent.mito"){
      max.y <- max.mt
    }
    plots.v <- list()
    densities.s <- list()
    plots.s <- list()
    for (j in names(sc10x)){
      sc10x.temp <- sc10x[[j]]
      plots.v[[j]] <- VlnPlot(object=sc10x.temp,features=i,pt.size=0.1,)+scale_x_discrete(labels=j)+scale_y_continuous(limits=c(0,max.y))+theme(legend.position="none",axis.text.x=element_text(hjust=0.5,angle=0))
      if (i %in% c("nFeature_RNA","percent.mito")){
        if (i == "nFeature_RNA"){
          cutoff.l <- thresh[[i]][[j]]$threshold[thresh[[i]][[j]]$method=="MinErrorI"]
          cutoff.h <- thresh[[i]][[j]]$threshold[thresh[[i]][[j]]$method=="RenyiEntropy"]
        } else {
          cutoff.l <- cutoff.l.mito[[j]]
          cutoff.h <- thresh[[i]][[j]]$threshold[thresh[[i]][[j]]$method=="Triangle"]
        }
        plots.v[[j]] <- plots.v[[j]]+geom_hline(yintercept=cutoff.l,size=0.5,color="red")+geom_hline(yintercept=cutoff.h,size=0.5,color="red")
        densities.s[[j]] <- density(sc10x.temp$nCount_RNA,sc10x.temp[[i]][,1],n=1000)
        plots.s[[j]] <- ggplotGrob(ggplot(data.frame(cbind(sc10x.temp$nCount_RNA,sc10x.temp[[i]][,1])))+geom_point(aes(x=X1,y=X2,color=densities.s[[j]]),size=0.1)+scale_x_continuous(limits=c(0,max.ct))+scale_y_continuous(limits=c(0,max.y))+scale_color_viridis(option="inferno")+labs(x="nCount",y=i,color="Density")+ggtitle(j)+cowplot::theme_cowplot()+theme(plot.title=element_text(size=7.5),axis.title=element_text(size=7.5),axis.text=element_text(size=5,angle=45),legend.position="bottom",legend.title=element_text(size=5,face="bold",vjust=1),legend.text=element_text(size=5,angle=45))+guides(color=guide_colourbar(barwidth=5,barheight=0.5))+geom_hline(yintercept=cutoff.l,size=0.1,color="red")+geom_hline(yintercept=cutoff.h,size=0.1,color="red"))
        cells.remove[[j]] <- c(cells.remove[[j]],rownames(sc10x[[j]][[i]])[sc10x[[j]][[i]][,1] < cutoff.l | sc10x[[j]][[i]][,1] > cutoff.h])
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      }
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      ggsave(paste0("./analysis/qc/Violin_qc.raw.",i,".",j,".eps"),plot=plots.v[[j]])
      if (i %in% c("nFeature_RNA","percent.mito")){
        ggsave(paste0("./analysis/qc/Plot_qc.raw.",i,".",j,".eps"),plot=plots.s[[j]])
      }
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    }
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  }
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  #Record cell/gene counts pre and post filtering
  counts.cell.raw <- list()
  counts.gene.raw <- list()
  sc10x.sub <- list()
  counts.cell.filtered <- list()
  counts.gene.filtered <- list()
  for (i in names(sc10x)){
    counts.cell.raw[i] <- ncol(GetAssayData(object=sc10x[[i]],slot="counts"))
    counts.gene.raw[i] <- nrow(GetAssayData(object=sc10x[[i]],slot="counts"))
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    sc10x.sub[[i]] <- subset(sc10x[[i]],cells=setdiff(colnames(sc10x[[i]]),cells.remove[[i]]))
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    counts.cell.filtered[i] <- ncol(GetAssayData(object=sc10x.sub[[i]],slot="counts"))
    counts.gene.filtered[i] <- nrow(GetAssayData(object=sc10x.sub[[i]],slot="counts"))
  }
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  #Plot filtered stats
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  max.ct <- 0
  max.ft <- 0
  max.mt <- 0
  for (i in names(sc10x)){
    if (max.ct < max(sc10x.sub[[i]][["nCount_RNA"]])){
      max.ct <- max(sc10x.sub[[i]][["nCount_RNA"]])
    }
    if (max.ft < max(sc10x.sub[[i]][["nFeature_RNA"]])){
      max.ft <- max(sc10x.sub[[i]][["nFeature_RNA"]])
    }
    if (max.mt < max(sc10x.sub[[i]][["percent.mito"]])){
      max.mt <- max(sc10x.sub[[i]][["percent.mito"]])
    }
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  }
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  max.ct <- max.ct*1.1
  max.ft <- max.ft*1.1
  max.mt <- max.mt*1.1
  for (i in c("nCount_RNA","nFeature_RNA","percent.mito")){
    max.y <- 0
    if (i == "nCount_RNA"){
      max.y <- max.ct
    } else if (i == "nFeature_RNA"){
      max.y <- max.ft
    } else if (i == "percent.mito"){
      max.y <- max.mt
    }
    plots.v <- list()
    densities.s <- list()
    plots.s <- list()
    for (j in names(sc10x.sub)){
      sc10x.temp <- sc10x.sub[[j]]
      plots.v[[j]] <- VlnPlot(object=sc10x.temp,features=i,pt.size=0.1,)+scale_x_discrete(labels=j)+scale_y_continuous(limits=c(0,max.y))+theme(legend.position="none",axis.text.x=element_text(hjust=0.5,angle=0))
      if (i %in% c("nFeature_RNA","percent.mito")){
        densities.s[[j]] <- density(sc10x.temp$nCount_RNA,sc10x.temp[[i]][,1],n=1000)
        plots.s[[j]] <- ggplotGrob(ggplot(data.frame(cbind(sc10x.temp$nCount_RNA,sc10x.temp[[i]][,1])))+geom_point(aes(x=X1,y=X2,color=densities.s[[j]]),size=0.1)+scale_x_continuous(limits=c(0,max.ct))+scale_y_continuous(limits=c(0,max.y))+scale_color_viridis(option="inferno")+labs(x="nCount",y=i,color="Density")+ggtitle(j)+cowplot::theme_cowplot()+theme(plot.title=element_text(size=7.5),axis.title=element_text(size=7.5),axis.text=element_text(size=5,angle=45),legend.position="bottom",legend.title=element_text(size=5,face="bold",vjust=1),legend.text=element_text(size=5,angle=45))+guides(color=guide_colourbar(barwidth=5,barheight=0.5)))
      }
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      ggsave(paste0("./analysis/qc/Violin_qc.filtered.",i,".",j,".eps"),plot=plots.v[[j]])
      if (i %in% c("nFeature_RNA","percent.mito")){
        ggsave(paste0("./analysis/qc/Plot_qc.filtered.",i,".",j,".eps"),plot=plots.s[[j]])
      }

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    }
  }
  
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  results <- list(
    sc10x=sc10x.sub,
    counts.cell.raw=counts.cell.raw,
    counts.gene.raw=counts.gene.raw,
    counts.cell.filtered=counts.cell.filtered,
    counts.gene.filtered=counts.gene.filtered
  )
  return(results)
}

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scThresh <- function(sc10x,feature,sub=FALSE){
  #Calculate thresholds and cutoffs
  
  #Inputs:
  #sc10x = Seruat object
  #feature = feature to threshold
  #sub = Subfolder to save output files
  
  #Outputs:
  #result = Threshold data
  
  
  #Make folders
  if (sub==FALSE){
    folder <- "./analysis/qc/cutoffs/"
  } else {
    folder <- paste0("./analysis/qc/cutoffs/",sub,"/")
    if (!dir.exists(folder)){
      dir.create(folder)
    }
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  }
  
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  #Calculate range of histogram based threholding and manually select methods for cutoffs
  scale <- list()
  scale.scaled <- list()
  h <- list()
  thresh <-list()
  cutoff.l <- list()
  cutoff.h <- list()
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  thresh_methods <- c("IJDefault","Huang","Huang2","Intermodes","IsoData","Li","Mean","MinErrorI","Moments","Otsu","Percentile","RenyiEntropy","Shanbhag","Triangle")
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  for (i in names(sc10x)){
    scale[[i]] <- data.frame(Score=sc10x[[i]][[feature]])
    colnames(scale[[i]]) <- "Score"
    scale[[i]] <- data.frame(Score=scale[[i]]$Score[!is.na(scale[[i]]$Score)])
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    scale.scaled[[i]] <- as.integer((scale[[i]]$Score-min(scale[[i]]$Score))/(max(scale[[i]]$Score)-min(scale[[i]]$Score))*360)
    #scale.scaled[[i]] <- as.integer(scales::rescale(scale[[i]]$Score,to=c(0,1))*360)
    h[[i]] <- hist(scale[[i]]$Score,breaks=100,plot=FALSE)
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    thresh[[i]] <- purrr::map_chr(thresh_methods, ~auto_thresh(scale.scaled[[i]], .)) %>% 
      tibble(method = thresh_methods, threshold = .)
    thresh[[i]]$threshold <- as.numeric(thresh[[i]]$threshold)
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    thresh[[i]]$threshold <- ((thresh[[i]]$threshold/360)*(max(scale[[i]]$Score)-min(scale[[i]]$Score)))+min(scale[[i]]$Score)
    #thresh[[i]] <- thresh[[i]] %>% mutate(threshold=(scales::rescale(as.numeric(threshold)/360,to=range(scale[[i]]$Score))))
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    postscript(paste0(folder,"Hist_qc.",i,".",feature,".eps"))
    plot(h[[i]],main=paste0("Histogram of ",feature," of sample ",i),xlab=feature)
    abline(v=thresh[[i]]$threshold)
    mtext(thresh[[i]]$method,side=1,line=2,at=thresh[[i]]$threshold,cex=0.5)
    dev.off()
  }
  
  
  return(thresh)
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}
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scCellCycle <- function(sc10x,sub=FALSE,sp="hu"){
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  #Runs Seurat based PCA analysis for cell cycle ID
  
  #Inputs:
  #sc10x = Seruat object
  #sub = Subfolder to save output files
  
  #Outputs:
  #results[1] = Seurat object
  #results[2] = s genes
  #results[3] = g2m genes
  
  #Make sub-folders if necessary
  if (sub==FALSE){
    folder <- "./analysis/qc/cellcycle/"
  } else {
    folder <- paste0("./analysis/qc/cellcycle/",sub,"/")
    if (!dir.exists(folder)){
      dir.create(folder)
    }}
  
  #score cell cycle
  genes.cc <- readLines(con="./genesets/regev_lab_cell_cycle_genes.txt")
  genes.s <- genes.cc[1:43]
  genes.g2m <- genes.cc[44:97]
  sc10x <- NormalizeData(object=sc10x,verbose=FALSE)
  sc10x <- ScaleData(object=sc10x,do.par=TRUE,num.cores=45,verbose=FALSE)
  sc10x <- CellCycleScoring(object=sc10x,s.features=genes.s,g2m.features=genes.g2m,set.ident=TRUE)
  
  #plot cell cycle specific genes
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  if (sp=="hu"){
    genes=c("PCNA","TOP2A","MCM6","MKI67")
    postscript(paste0(folder,"Violin_cc.Raw.eps"))
    plot <- VlnPlot(object=sc10x,features=genes,ncol=2,pt.size=1)
    plot(plot)
    dev.off()
  }
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  # sc10x <- RunPCA(object=sc10x,features=c(genes.s,genes.g2m),npcs=2,verbose=FALSE)
  # postscript(paste0(folder,"PCA_cc.Raw.eps"))
  # plot <- DimPlot(object=sc10x,reduction="pca")
  # plot(plot)
  # dev.off()
  # gc()
  # sc10x <- ScaleData(object=sc10x,vars.to.regress=c("S.Score","G2M.Score"),do.par=TRUE,num.cores=45,verbose=TRUE)
  # gc()
  # sc10x <- RunPCA(object=sc10x,features=c(genes.s,genes.g2m),npcs=2,verbose=FALSE)
  # postscript(paste0(folder,"PCA_cc.Norm.eps"))
  # plot <- DimPlot(object=sc10x,reduction="pca")
  # plot(plot)
  # dev.off()
  
  results <- list(
    sc10x=sc10x,
    genes.s=genes.s,
    genes.g2m=genes.g2m
  )
  return(results)
}


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scPC <- function(sc10x,pc=50,hpc=0.9,file="pre.stress",print="tsne"){
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  #Scale Seurat object & calculate # of PCs to use
  
  #Inputs:
  #sc10x = Seruat object
  #pc = number of PCs to cacluate
  #hpc = max variance cutoff for PCs to use"
  #file = file for output
  
  #Outputs:
  #result[1] = Seurat object
  #result[2] = # of PCs to use
  
  #Run PCA
  Idents(object=sc10x) <- "ALL"
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  sc10x <- RunPCA(object=sc10x,npcs=pc,verbose=FALSE,assay="integrated")
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  #Calculate PCs to use
  pc.use <- sc10x[["pca"]]@stdev^2
  pc.use <- pc.use/sum(pc.use)
  pc.use <- cumsum(pc.use)
  pc.use <- min(which(pc.use>=hpc))
  
  postscript(paste0("./analysis/qc/Plot_PCElbow_",file,".eps"))
  plot <- ElbowPlot(object=sc10x,ndims=pc)
  plot <- plot+geom_vline(xintercept=pc.use,size=1,color="red")
  plot(plot)
  dev.off()
  
  results <- list(
    sc10x=sc10x,
    pc.use=pc.use
  )
  return(results)
}


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scCCA <-  function(sc10x.l){
  for (i in 1:length(sc10x.l)){
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    #sc10x.l[[i]] <- NormalizeData(sc10x.l[[i]],verbose=FALSE)
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    gc()
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    #sc10x.l[[i]] <- ScaleData(sc10x.l[[i]],vars.to.regress=c("nFeature_RNA","percent.mito"),verbose = FALSE)
    sc10x.l[[i]] <- SCTransform(sc10x.l[[i]],vars.to.regress=c("nFeature_RNA","percent.mito"),verbose=FALSE,assay="RNA")
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    gc()
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    #sc10x.l[[i]] <- FindVariableFeatures(sc10x.l[[i]],selection.method="vst",nfeatures=2000,verbose=FALSE)
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  }
  
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  sc10x.features <- SelectIntegrationFeatures(object.list=sc10x.l,nfeatures=3000)
  sc10x.l <- PrepSCTIntegration(object.list=sc10x.l,anchor.features=sc10x.features,verbose=FALSE)
  sc10x.anchors <- FindIntegrationAnchors(object.list=sc10x.l, normalization.method="SCT",anchor.features=sc10x.features,verbose=FALSE)
  sc10x <- IntegrateData(anchorset=sc10x.anchors,normalization.method="SCT",verbose=FALSE)
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  #sc10x <- FindIntegrationAnchors(object.list=sc10x.l,dims=1:30,scale=FALSE)
  #sc10x <- IntegrateData(anchorset=sc10x,dims=1:30)
  
  #gc()
  #sc10x <- ScaleData(object=sc10x,do.par=TRUE,num.cores=45,verbose=FALSE,assay="integrated")
  #gc()
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  gc()
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  sc10x <- SCTransform(sc10x,vars.to.regress=c("nFeature_RNA","percent.mito"),verbose=FALSE,return.only.var.genes=FALSE,assay="RNA")
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  gc()
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  return(sc10x)
}


scCluster <- function(sc10x,res=0.1,red="pca",dim,print="tsne",folder=FALSE){
  #Cluster Seurat object and produce visualizations
  
  #Inputs:
  #sc10x = Seruat object
  #res = resolution to calculate clustering
  #red = rediction type to use for clustering
  #dim = number of dimentions to use for display
  #print = dimentionality reduction to use for display
  #folder = folder for output
  
  #Outputs:
  #result = Seurat object
  
  #Create subfolder if necessary
  if (folder==FALSE){
    sub <- ""
  } else {
    if (!dir.exists(paste0("./analysis/vis/",folder))){
      dir.create(paste0("./analysis/vis/",folder))
    }
    sub <- paste0(folder,"/")
    
  }
  
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  DefaultAssay(sc10x) <- "integrated"

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  #Calculste Vis
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  sc10x <- RunTSNE(sc10x,dims=1:dim,reduction="pca",assay="integrated")
  sc10x <- RunUMAP(sc10x,dims=1:dim,reduction="pca",assay="integrated")
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  sc10x <- FindNeighbors(sc10x,reduction=red,verbose=FALSE)
  
  for (i in res){
    sc10x <- FindClusters(sc10x,resolution=i,verbose=FALSE)
    
    plot1 <- DimPlot(sc10x,reduction="pca",label=TRUE,repel=TRUE)+theme(legend.position="none")
    plot2 <- DimPlot(sc10x,reduction="tsne",label=TRUE,repel=TRUE)+theme(legend.position="none")
    plot3 <- DimPlot(sc10x,reduction="umap",label=TRUE,repel=TRUE)+theme(legend.position="none")
    
    if (print=="tsne"){
      postscript(paste0("./analysis/vis/",sub,"tSNE_",i,".eps"))
      print(print2)
      dev.off()
    } else if (print=="umap"){
      postscript(paste0("./analysis/vis/",sub,"UMAP_",i,".eps"))
      print(print3)
      dev.off()
    } else if (print=="2"){
      plot2 <- plot2+theme(legend.position="none")
      plot3 <- plot3+theme(legend.position="none")
      postscript(paste0("./analysis/vis/",sub,"2Vis_",i,".eps"))
      grid.arrange(plot2,plot3,ncol=1)
      dev.off()
    } else if (print=="3"){
      plot1 <- plot1+theme(legend.position="none")
      plot2 <- plot2+theme(legend.position="none")
      plot3 <- plot3+theme(legend.position="none")
      postscript(paste0("./analysis/vis/",sub,"3Vis_",i,".eps"))
      grid.arrange(plot1,plot2,plot3,ncol=1)
      dev.off()
    }}
  
  plot1 <- DimPlot(sc10x,reduction="pca",group.by="samples")
  plot2 <- DimPlot(sc10x,reduction="tsne",group.by="samples")
  plot3 <- DimPlot(sc10x,reduction="umap",group.by="samples")
  legend <- cowplot::get_legend(plot1)
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  if (print=="tsne"){
    postscript(paste0("./analysis/vis/",sub,"tSNE_samples.eps"))
    grid.arrange(plot2,legend,ncol=1)
    dev.off()
  } else if (print=="umap"){
    postscript(paste0("./analysis/vis/",sub,"UMAP_samples.eps"))
    grid.arrange(plot3,legend,ncol=1)
    dev.off()
  } else if (print=="2"){
    plot2 <- plot2+theme(legend.position="none")
    plot3 <- plot3+theme(legend.position="none")
    postscript(paste0("./analysis/vis/",sub,"2Vis_samples.eps"))
    grid.arrange(plot2,plot3,legend,ncol=1)
    dev.off()
  } else if (print=="3"){
    plot1 <- plot1+theme(legend.position="none")
    plot2 <- plot2+theme(legend.position="none")
    plot3 <- plot3+theme(legend.position="none")
    postscript(paste0("./analysis/vis/",sub,"3Vis_samples.eps"))
    grid.arrange(plot1,plot2,plot3,legend,ncol=1)
    dev.off()
  }
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  DefaultAssay(sc10x) <- "SCT"
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  return(sc10x)
}


scScore <- function(sc10x,score,geneset,cut.pt=0.9,anchor=FALSE){
  #Runs custom PCA analysis for stress ID
  
  #Inputs:
  #sc10x = Seruat object
  #score = name of geneset for scoring
  #geneset = geneset to use for ID
  #cut.pt = % of cells to keep
  
  #Outputs:
  #results[1] = Seurat object (original + score)
  #results[2] = Seurat object (negatively filtered)
  #results[3] = Seurat object (positively filtered)
  
  #Make subdirectory
  if (!dir.exists(paste0("./analysis/score_id/",score))){
    dir.create(paste0("./analysis/score_id/",score))
  }
  if (!dir.exists(paste0("./analysis/vis/",score))){
    dir.create(paste0("./analysis/vis/",score))
  }
  
  #Score geneset
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  sc10x <- AddModuleScore(object=sc10x,features=geneset,name=score,assay="SCT")
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  Idents(object=sc10x) <- paste0(score,"1")
  
  #CDF
  cdf <- ecdf(as.numeric(levels(sc10x)))
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  if (cut.pt == "renyi"){
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    thresh <- list()
    thresh[["all"]] <- scThresh(list(all=sc10x),feature=paste0(score,"1"),sub=score)
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    cut.x <- thresh$all$all$threshold[thresh$all$all$method=="RenyiEntropy"]
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  } else {
    cut.x <- quantile(cdf,probs=cut.pt)
    cut.x <- unname(cut.x)
  }
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  postscript(paste0("./analysis/score_id/",score,"/CDF_",score,".eps"))
  plot(cdf,main=paste0("Cumulative Distribution of ",score," Score"),xlab=paste0(score," Score"),ylab="CDF")
  abline(v=cut.x,col="red")
  dev.off()  
  
  #KDE
  postscript(paste0("./analysis/score_id/",score,"/Histo_",score,".eps"))
  plot(ggplot(data.frame(Score=as.numeric(levels(sc10x))),aes(x=Score))+geom_histogram(bins=100,aes(y=..density..))+geom_density()+geom_vline(xintercept=cut.x,size=1,color="red")+ggtitle(paste0(score," Score"))+cowplot::theme_cowplot())
  dev.off()
  
  Idents(object=sc10x) <- "ALL"
  predicate <- paste0(score,"1 >= ",cut.x)
  Idents(object=sc10x, cells = WhichCells(object=sc10x,expression= predicate)) <- score
  sc10x[[score]] <- Idents(object=sc10x)
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  Idents(sc10x) <- score
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  sc10x.negative <- subset(x=sc10x,idents="ALL")
  sc10x.positive <- subset(x=sc10x,idents=score)
  
  #Generate plots
  postscript(paste0("./analysis/vis/",score,"/3Vis_",score,".eps"))
  plot1 <- DimPlot(sc10x,reduction="pca",label=TRUE,repel=TRUE)+theme(legend.position="none")+ggtitle("ALL")
  plot2 <- DimPlot(sc10x.negative,reduction="pca",label=TRUE,repel=TRUE)+theme(legend.position="none")
  plot3 <- DimPlot(sc10x.positive,reduction="pca",label=TRUE,repel=TRUE)+theme(legend.position="none")
  plot4 <- DimPlot(sc10x,reduction="tsne",label=TRUE,repel=TRUE)+theme(legend.position="none")+ggtitle("Negative")
  plot5 <- DimPlot(sc10x.negative,reduction="tsne",label=TRUE,repel=TRUE)+theme(legend.position="none")
  plot6 <- DimPlot(sc10x.positive,reduction="tsne",label=TRUE,repel=TRUE)+theme(legend.position="none")
  plot7 <- DimPlot(sc10x,reduction="umap",label=TRUE,repel=TRUE)+theme(legend.position="none")+ggtitle("Positive")
  plot8 <- DimPlot(sc10x.negative,reduction="umap",label=TRUE,repel=TRUE)+theme(legend.position="none")
  plot9 <- DimPlot(sc10x.positive,reduction="umap",label=TRUE,repel=TRUE)+theme(legend.position="none")
  grid.arrange(plot1,plot2,plot3,plot4,plot5,plot6,plot7,plot8,plot9,ncol=3)
  dev.off()
  
  #Generate violin plot of gene exvpression
  if (anchor!=FALSE){
    postscript(paste0("./analysis/score_id/",score,"/Violin_",score,".eps"))
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    plot <- VlnPlot(object=sc10x,features=anchor,pt.size=0.1,assay="SCT")
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    plot(plot)
    dev.off()
  }
  
  results <- list(
    sc10x <- sc10x,
    sc10x.negative <- sc10x.negative,
    sc10x.positive <- sc10x.positive
  )
  return(results)
}


scQuSAGE <- function(sc10x,gs,save=FALSE,type,id,ds=0,nm="pops",print="tsne"){
  #Runs QuSAGE
  
  #Inputs:
  #sc10x = Seruat object
  #gs = geneset to use for correlation
  #save = save ID
  #type = type of qusage to run (id: create ID's based on run, sm: cor only using small genesets, lg: cor only with large genesets)
  #id = ident to use
  #nm = name of test
  #print = dimentionality reduction to use for display
  
  #Outputs:
  #results[1] = Seurat object
  #results[2] = correlation table
  #results[3] = correlation results
  
  if (!dir.exists(paste0("./analysis/cor/",nm))){
    dir.create(paste0("./analysis/cor/",nm))
  }
  if (!dir.exists(paste0("./analysis/cor/",nm,"/geneset"))){
    dir.create(paste0("./analysis/cor/",nm,"/geneset"))
  }
  if (!dir.exists(paste0("./analysis/cor/",nm,"/cluster"))){
    dir.create(paste0("./analysis/cor/",nm,"/cluster"))
  }
  if (!dir.exists(paste0("./analysis/vis/",nm))){
    dir.create(paste0("./analysis/vis/",nm))
  }
  
  Idents(object=sc10x) <- id
  number.clusters <- length(unique(levels(x=sc10x)))
  
  labels <- paste0("Cluster_",as.vector(Idents(object=sc10x)))
  
  cell.sample <- NULL
  for (i in unique(labels)){
    cell <- WhichCells(sc10x,ident=sub("Cluster_","",i))
    if (length(cell)>ds & ds!=0){
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      set.seed(71682)
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      rnd <- sample(1:length(cell),ds)
      cell <- cell[rnd]
    }
    cell.sample <- c(cell.sample,cell)
  }
  data <- as.data.frame(as.matrix(GetAssayData(sc10x[,colnames(sc10x) %in% cell.sample])))
  labels <- labels[colnames(sc10x) %in% cell.sample]
  groups <- sort(unique(labels))
  
  col <- hcl(h=(seq(15,375-375/length(groups),length=length(groups))),c=100,l=65)
  
  #Make labels for QuSAGE
  clust <- list()
  clust.comp <- list()
  for (i in groups){
    t <- labels
    t[t != i] <- "REST"
    clust[i] <- list(i=t)
    rm(t)
    clust.comp[i] <- paste0(i,"-REST")
  }
  
  #Run QuSAGE
  for (i in groups){
    assign(paste0("results.",i),qusage(data,unlist(clust[i]),unlist(clust.comp[i]),gs))
  }
  
  #Generate ID table
  results.cor <- NULL
  results.cor <- qsTable(get(paste0("results.",groups[1])),number=length(gs))
  results.cor$Cluster <- groups[1]
  for (i in groups[-1]){
    qs <- qsTable(get(paste0("results.",i)),number=length(gs))
    qs$Cluster <- i
    results.cor <- rbind(results.cor,qs)
  }
  results.cor <- results.cor[,-3]
  rownames(results.cor) <- NULL
  
  results.clust.id <- NULL
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  #if (max(results.cor[results.cor[,4]==groups[1] & results.cor[,3]<=0.05,][,2],na.rm=TRUE)>=0){
  #  results.clust.id <- results.cor[results.cor[,4]==groups[1] & results.cor[,3]<=0.05,][which.max(results.cor[results.cor[,4]==groups[1] & results.cor[,3]<=0.05,][,2]),]
  if (max(results.cor[results.cor[,4]==groups[1],][,2],na.rm=TRUE)>=0){
    results.clust.id <- results.cor[results.cor[,4]==groups[1],][which.max(results.cor[results.cor[,4]==groups[1],][,2]),]
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  } else {
    results.clust.id$pathway.name <- "Unknown"
    results.clust.id$log.fold.change <- 0
    results.clust.id$FDR <- 0
    results.clust.id$Cluster <- groups[1]
    results.clust.id <- as.data.frame(results.clust.id)
  }
  for (i in groups[-1]){
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    #if (max(results.cor[results.cor[,4]==i & results.cor[,3]<=0.05,][,2],na.rm=TRUE)>=0){
    #  results.clust.id <- rbind(results.clust.id,results.cor[results.cor[,4]==i & results.cor[,3]<=0.05,][which.max(results.cor[results.cor[,4]==i & results.cor[,3]<=0.05,][,2]),])
    if (max(results.cor[results.cor[,4]==i,][,2],na.rm=TRUE)>=0){
      results.clust.id <- rbind(results.clust.id,results.cor[results.cor[,4]==i,][which.max(results.cor[results.cor[,4]==i,][,2]),])
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    } else {
      results.clust.id <- rbind(results.clust.id,data.frame(pathway.name="Unknown",log.fold.change=0,FDR=0,Cluster=i))
    }}
  rownames(results.clust.id) <- NULL
  
  #Determine axes for correlation plots
  max.x.rg <- 0
  min.x.rg <- 0
  max.y.rg <- 0
  for (i in groups){
    qs <- get(paste0("results.",i))
    if (max(qs$path.mean)>max.x.rg){
      max.x.rg <- max(qs$path.mean)
    }
    if (min(qs$path.mean)<min.x.rg){
      min.x.rg <- min(qs$path.mean)
    }
    if (max(qs$path.PDF)>max.y.rg){
      max.y.rg <- max(qs$path.PDF)
    }}
  if (type=="sm"){
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    #Plot correlation plots by geneset
    for (i in 1:length(gs)){
      postscript(paste0("./analysis/cor/",nm,"/geneset/QuSAGE_",nm,".",names(gs)[i],".eps"))
      for (j in groups){
        qs <- get(paste0("results.",j))
        if (j==groups[1]){
          plotDensityCurves(qs,path.index=i,col=col[match(j,groups)],main=names(gs)[i],xlim=c(min.x.rg-0.05,max.x.rg+0.05),ylim=c(0,50*ceiling(max.y.rg/50)),xlab="Gene Set Activation",lwd=5,cex.main=2.5,cex.axis=1.5,cex.lab=2)
        } else {
          plotDensityCurves(qs,path.index=i,add=TRUE,col=col[match(j,groups)],lwd=5)
        }}
      legend("topright",col=col,legend=groups,lty=1,lwd=5,cex=2,ncol=1,bty="n",pt.cex=2)
      box(lwd=5)
      dev.off()
    }
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    #Plot correlation plots by cluster
    for (i in groups){
      qs <- get(paste0("results.",i))
      postscript(paste0("./analysis/cor/",nm,"/cluster/QuSAGE_",nm,"_",i,".eps"))
      for (j in 1:length(gs)){
        if (j==1){
          plotDensityCurves(qs,path.index=j,col=viridis(length(gs))[j],main=i,xlim=c(min.x.rg-0.05,max.x.rg+0.05),ylim=c(0,50*ceiling(max.y.rg/50)),xlab="Gene Set Activation",lwd=5,cex.main=2.5,cex.axis=1.5,cex.lab=2)
        } else {
          plotDensityCurves(qs,path.index=j,add=TRUE,col=viridis(length(gs))[j],lwd=5)
        }}
      legend("topright",col=viridis(length(gs)),legend=names(gs),lty=1,lwd=5,cex=1,ncol=2,bty="n",pt.cex=2)
      box(lwd=5)
      dev.off()
    }} else {
      for (i in groups){
        qs <- get(paste0("results.",i))
        postscript(paste0("./analysis/cor/",nm,"/cluster/QuSAGE_",nm,"_",i,".eps"))
        plotCIs(qs,path.index=1:numPathways(qs),cex.lab=1.5)
        dev.off()
      }}
  
  if (save==TRUE){
    merge.cluster <- NULL
    for (i in groups){
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      #if (max(qsTable(get(paste0("results.",i)),number=length(gs))[qsTable(get(paste0("results.",i)),number=length(gs))[,4]<=0.05,][,2],na.rm=TRUE)>=0){
      #  sc10x<-eval(parse(text=paste0("RenameIdents(object=sc10x,'",sub("Cluster_","",i),"' = '",qsTable(get(paste0("results.",i)),number=length(gs))[qsTable(get(paste0("results.",i)),number=length(gs))[2]==max(qsTable(get(paste0("results.",i)),number=length(gs))[qsTable(get(paste0("results.",i)),number=length(gs))[,4]<=0.05,][,2],na.rm=TRUE)][1],"')")))
      if (max(qsTable(get(paste0("results.",i)),number=length(gs))[,2],na.rm=TRUE)>=0){
        sc10x<-eval(parse(text=paste0("RenameIdents(object=sc10x,'",sub("Cluster_","",i),"' = '",qsTable(get(paste0("results.",i)),number=length(gs))[qsTable(get(paste0("results.",i)),number=length(gs))[2]==max(qsTable(get(paste0("results.",i)),number=length(gs))[,2],na.rm=TRUE)][1],"')")))
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      } else {
        sc10x<-eval(parse(text=paste0("RenameIdents(object=sc10x,'",sub("Cluster_","",i),"' = 'Unknown')")))
      }}
    sc10x[[nm]] <- Idents(object=sc10x)
  }
  
  plot1 <- DimPlot(sc10x,reduction="pca",label=TRUE,repel=TRUE)+theme(legend.position="none")
  plot2 <- DimPlot(sc10x,reduction="tsne",label=TRUE,repel=TRUE)+theme(legend.position="none")
  plot3 <- DimPlot(sc10x,reduction="umap",label=TRUE,repel=TRUE)+theme(legend.position="none")
  if (print=="tsne"){
    postscript(paste0("./analysis/vis/",nm,"/tSNE_",id,"_",nm,".eps"))
    print(print2)
    dev.off()
  } else if (print=="umap"){
    postscript(paste0("./analysis/vis/",nm,"/UMAP_",id,"_",nm,".eps"))
    print(print3)
    dev.off()
  } else if (print=="2"){
    postscript(paste0("./analysis/vis/",nm,"/2Vis_",id,"_",nm,".eps"))
    grid.arrange(plot2,plot3,ncol=1)
    dev.off()
  } else if (print=="3"){
    postscript(paste0("./analysis/vis/",nm,"/3Vis_",id,"_",nm,".eps"))
    grid.arrange(plot1,plot2,plot3,ncol=1)
    dev.off()
  }
  
  results <- list(
    sc10x=sc10x,
    results.cor=results.cor,
    results.clust.id=results.clust.id
  )
  names(results)=c("sc10x",paste0("results.cor.",nm),paste0("results.clust.",nm,".id"))
  return(results)
}
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scShinyOutput <- function(){
  for (i in c("pca","tsne","umap")){
    dr <- Embeddings(sc10x,i)
    if (i != "pca"){
      colnames(dr) <- c(paste0(toupper(i),"-",1:2))
    } else {
      dr <- dr[,1:10]
      colnames(dr) <- c(paste0(toupper(i),"-",1:10))
    }
    dr <- cbind(dr,Barcode=rownames(dr))
    dr <- dr[,c(3,1,2)]
    dr <- as.data.frame(dr,row.names=FALSE)
    if (i != "pca"){
      write_csv(dr,paste0("./analysis/shiny/raw/outs/analysis/",i,"/2_components/projection.csv"),col_names=TRUE)
    } else {
      write_csv(dr,paste0("./analysis/shiny/raw/outs/analysis/",i,"/10_components/projection.csv"),col_names=TRUE)
    }
  }
  for (i in c("samples",paste0("integrated_snn_res.",res))){
    if (!dir.exists(paste0("./analysis/shiny/raw/outs/analysis/clustering/",gsub("integrated_snn_res.","res_",i)))){
      dir.create(paste0("./analysis/shiny/raw/outs/analysis/clustering/",gsub("integrated_snn_res.","res_",i)))
    }
    clust <- as.matrix(sc10x[[i]])
    colnames(clust) <- "Cluster"
    clust <- cbind(clust,Barcode=rownames(clust))
    clust <- clust[,c(2,1)]
    clust <- as.data.frame(clust,row.names=FALSE)
    write_csv(clust,paste0("./analysis/shiny/raw/outs/analysis/clustering/",gsub("integrated_snn_res.","res_",i),"/clusters.csv"),col_names=TRUE)
  }
  
  #Idents(sc10x) <- "integrated_snn_res.0.1"
  #deg <- FindAllMarkers(sc10x,assay="SCT",slot="scale.data",logfc.threshold=0,test.use="MAST")
}