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Alyssa Salazar
AlyssaS/
RenalTumor
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[40a513]: / ATAC / AnalysisPipeline / 4.2.peakAnnotation.genomicLocation.R

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#' @description: peak annotation and enrichment analysis



##reference：

#https://hbctraining.github.io/Intro-to-ChIPseq/lessons/12_functional_analysis.html

#http://bioconductor.org/packages/devel/bioc/vignettes/ChIPseeker/inst/doc/ChIPseeker.html

library(Signac)

library(Seurat)

library(ggpubr)

library(GenomicRanges)

library(tibble)

library(dplyr)

set.seed(101) 



setwd(dir = "/data/active_data/lzl/RenalTumor-20200713/DataAnalysis-20210803/scATAC")

scATAC.data <- readRDS("scATAC.data.pro.rds")

DefaultAssay(scATAC.data) <- "Peaks"



## loading packages

library(ChIPseeker)

library(TxDb.Hsapiens.UCSC.hg38.knownGene)

txdb <- TxDb.Hsapiens.UCSC.hg38.knownGene

library(clusterProfiler)

library(org.Hs.eg.db)



#####################################All Peak Annotation

peakSet <- GetAssayData(scATAC.data, slot = "data") 

All.gr <- StringToGRanges(rownames(peakSet))

#TSS (transcription start site) region, by default TSS is defined from -3kb to +3kb

All.peakAnno <- annotatePeak(All.gr, tssRegion=c(-3000, 3000), TxDb = txdb, annoDb="org.Hs.eg.db")

saveRDS(All.peakAnno, file = "4.Peak/peak.annotation.ChIPseeker.rds")

peakAnno.info <- All.peakAnno@anno

peakAnno.info$annotation <- gsub(" .*", "", peakAnno.info$annotation)

peakAnno.info$annotation <- gsub("'", "_UTR", peakAnno.info$annotation)

peak.info <- data.frame(peaks = rownames(Motifs(scATAC.data)@data),

                        originType = All.peakAnno@anno$annotation,

                        peakType = peakAnno.info$annotation, 

                        distanceToTSS = peakAnno.info$distanceToTSS,

                        SYMBOL = peakAnno.info$SYMBOL)

saveRDS(peak.info, file = "4.Peak/peak.annotation.simple.ChIPseeker.rds")

promoter <- getPromoters(TxDb=txdb, upstream=3000, downstream=3000)

tagMatrix <- getTagMatrix(All.gr, windows=promoter)



pdf("4.Peak/Annotation/All.peakAnnotation.pdf")

plotAnnoPie(All.peakAnno)

plotAnnoBar(All.peakAnno)

vennpie(All.peakAnno) #some annotation overlap

upsetplot(All.peakAnno, vennpie=TRUE)

#Visualize distribution of TF-binding loci relative to TSS

plotDistToTSS(All.peakAnno, title="Distribution of transcription factor-binding loci\nrelative to TSS")

tagHeatmap(tagMatrix, xlim=c(-3000, 3000), color="#4DBBD5")

plotAvgProf(tagMatrix, xlim=c(-3000, 3000), xlab="Genomic Region (5'->3')", ylab = "Read Count Frequency")

plotAvgProf(tagMatrix, xlim=c(-3000, 3000), conf = 0.95, resample = 1000)

dev.off()



#######################################annotated the cell-type specific peaks

cellType.sig.pos.DARs <- readRDS("4.Peak/cellType.sig.pos.DARs.rds")

cellType.sig.pos.gr <- StringToGRanges(unique(cellType.sig.pos.DARs$genomicRegion)) # 8797 DAR peaks

DAR.Anno <- annotatePeak(cellType.sig.pos.gr, tssRegion=c(-3000, 3000), TxDb = txdb, annoDb="org.Hs.eg.db")



idents <- unique(cellType.sig.pos.DARs$cellType)

cellType.gr.list <- lapply(idents, function(x){

  idx <- which(cellType.sig.pos.DARs$cellType == x)

  gr <- StringToGRanges(cellType.sig.pos.DARs$genomicRegion[idx])

})

names(cellType.gr.list) <- idents



#annotated DAR for each cell type

DAR.anno.list <- lapply(cellType.gr.list, function(x){

    DAR.cell <- annotatePeak(x, tssRegion=c(-3000, 3000), TxDb = txdb, annoDb="org.Hs.eg.db")

    return(DAR.cell)

})

promoter <- getPromoters(TxDb=txdb, upstream=3000, downstream=3000)

tagMatrix <- getTagMatrix(cellType.sig.pos.gr, windows=promoter)



pdf("4.Peak/Annotation/cellType.DAR.annotation.pdf")

tagHeatmap(tagMatrix, xlim=c(-3000, 3000), color="#4DBBD5")

plotAvgProf(tagMatrix, xlim=c(-3000, 3000), xlab="Genomic Region (5'->3')", ylab = "Read Count Frequency")

plotAvgProf(tagMatrix, xlim=c(-3000, 3000), conf = 0.95, resample = 1000)

plotAnnoPie(DAR.Anno)

plotAnnoBar(DAR.Anno)

vennpie(DAR.Anno) #some annotation overlap

upsetplot(DAR.Anno, vennpie=TRUE)



#Visualize distribution of TF-binding loci relative to TSS

plotDistToTSS(DAR.Anno, title="Distribution of transcription factor-binding loci\nrelative to TSS")



plotAnnoBar(DAR.anno.list)

plotDistToTSS(DAR.anno.list)

dev.off()



############################# write DARs to bed files

# write all peaks to file to serve as background set:

DefaultAssay(scATAC.data) <- "Peaks"

peak_ranges <- StringToGRanges(rownames(scATAC.data)) %>% sort

write.table(as.data.frame(peak_ranges)[,1:3], file='4.Peak/bed/allPeaks.bed', row.names=F, col.names=F, sep='\t', quote=F)



########### each cell type

All.DARs <- readRDS("4.Peak/cellType.all.DARs.rds") # 224807 region

idents <- levels(All.DARs$cellType)

res < sapply(idents, function(x){

  cat("writing ", x, "\n")

  DARs.up <- All.DARs %>% dplyr::filter(cellType == x & avg_log2FC >= 0.25 & p_val_adj < 0.05)

  DARs.down <- All.DARs %>% dplyr::filter(cellType == x & avg_log2FC < -0.25 & p_val_adj < 0.05)



  if(nrow(DARs.up) >= 25){

    DARs.up.info <- peakAnno.info.frame[match(DARs.up$genomicRegion, peakAnno.info.frame$id),]

    # distal

    distal.peak.up <- DARs.up.info[which(DARs.up.info$annotation == "Distal"),]

    write.table(distal.peak.up[,1:3], file = paste0('4.Peak/bed/', x,'_up_distalPeaks.bed'), row.names=F, col.names=F, sep='\t', quote=F)

    cat("Distal Up: ", nrow(distal.peak.up), "\n")



    # proximal

    proximal.peak.up <- DARs.up.info[which(DARs.up.info$annotation != "Distal"),]

    write.table(proximal.peak.up[,1:3], file = paste0('4.Peak/bed/', x,'_up_proximalPeaks.bed'), row.names=F, col.names=F, sep='\t', quote=F)

    cat("proximal Up: ", nrow(proximal.peak.up), "\n")

  }



  if(nrow(DARs.down) >= 25){

    DARs.down.info <- peakAnno.info.frame[match(DARs.down$genomicRegion, peakAnno.info.frame$id),]

    # distal

    distal.peak.down <- DARs.down.info[which(DARs.down.info$annotation == "Distal"),]

    write.table(distal.peak.down[,1:3], file = paste0('4.Peak/bed/', x,'_down_distalPeaks.bed'), row.names=F, col.names=F, sep='\t', quote=F)

    cat("Distal Down: ", nrow(distal.peak.down), "\n")



    # proximal

    proximal.peak.down <- DARs.down.info[which(DARs.down.info$annotation != "Distal"),]

    write.table(proximal.peak.down[,1:3], file = paste0('4.Peak/bed/', x,'_down_proximalPeaks.bed'), row.names=F, col.names=F, sep='\t', quote=F)

    cat("proximal Down: ", nrow(proximal.peak.down), "\n")

  }

  return("Finish!")

})



##rGREAT enrichment analysis

source(file = "/home/longzhilin/Analysis_Code/PathwayEnrichment/Great.enrich.R")



idents <- levels(scATAC.data)

bg <- read.table("4.Peak/bed/allPeaks.bed", sep = "\t", stringsAsFactors = F)



pdf("4.Peak/bed/GREAT.enrich.pdf")

res <- lapply(idents, function(x){



  ## up regulate

  up.distal.bed <- tryCatch({

    read.table(paste0("4.Peak/bed/", x, "_up_distalPeaks.bed"), sep = "\t", stringsAsFactors = F)}, error = function(e){return(NULL)})

  if(!is.null(up.distal.bed)){

    cat("Analysis: ", x, " up distal region...\n")

    up_distalPeaks_enrich <- Great.enrich(gr = up.distal.bed, bg = bg, title = paste0(x, "_up_distalPeaks"), Type = "GO Biological Process")

  }else{

    up_distalPeaks_enrich <- NULL

  }

  up.proximal.bed <- tryCatch({

    read.table(paste0("4.Peak/bed/", x, "_up_proximalPeaks.bed"), sep = "\t", stringsAsFactors = F)}, error = function(e){return(NULL)})

  if(!is.null(up.proximal.bed)){

    cat("Analysis: ", x, " up proximal region...\n")  

    up_proximalPeaks_enrich <- Great.enrich(gr = up.proximal.bed, bg = bg, title = paste0(x, "_up_proximalPeaks"), Type = "GO Biological Process")

  }else{

    up_proximalPeaks_enrich <- NULL

  }



  ## down regulate

  down.distal.bed <- tryCatch({

    read.table(paste0("4.Peak/bed/", x, "_down_distalPeaks.bed"), sep = "\t", stringsAsFactors = F)}, error = function(e){return(NULL)})

  if(!is.null(down.distal.bed)){

    cat("Analysis: ", x, " down distal region...\n")

    down_distalPeaks_enrich <- Great.enrich(gr = down.distal.bed, bg = bg, title = paste0(x, "_down_distalPeaks"), Type = "GO Biological Process")

  }else{

    down_distalPeaks_enrich <- NULL

  }

  down.proximal.bed <- tryCatch({

    read.table(paste0("4.Peak/bed/", x, "_down_proximalPeaks.bed"), sep = "\t", stringsAsFactors = F)}, error = function(e){return(NULL)})

  if(!is.null(down.proximal.bed)){

    cat("Analysis: ", x, " down proximal region...\n")  

    down_proximalPeaks_enrich <- Great.enrich(gr = down.proximal.bed, bg = bg, title = paste0(x, "_down_proximalPeaks"), Type = "GO Biological Process")

  }else{

    down_proximalPeaks_enrich <- NULL

  }

  return(list(up_distalPeaks_enrich = up_distalPeaks_enrich, up_proximalPeaks_enrich = up_proximalPeaks_enrich, 

              down_distalPeaks_enrich = down_distalPeaks_enrich, down_proximalPeaks_enrich = down_proximalPeaks_enrich))

})

dev.off()

names(res) <- idents

saveRDS(res, file = "4.Peak/bed/GREAT.enrichment.rds")

library(openxlsx)

write.xlsx(res$Tumor, file = "4.Peak/bed/GREAT.Tumor.xlsx", sheetName = names(res$Tumor), rowNames = F)



####plot --- tumor.up

tumor.up.proximal <- res$Tumor$up_proximalPeaks_enrich

tumor.up.distal <- res$Tumor$up_distalPeaks_enrich

pdf("4.Peak/bed/GREAT.Tumor.selection.pdf", height = unit(5, "inches"))

p <- ggbarplot(tumor.up.proximal, x = "wrap", y = "Hyper_Fold_Enrichment", fill = "grey", color = "grey", width = 0.4, ylab = "Fold Enrichment", xlab = "", orientation = "horiz", sort.val = c("asc"))+theme(legend.position="none") 

print(p)

p <- ggbarplot(tumor.up.distal, x = "wrap", y = "Hyper_Fold_Enrichment", fill = "grey", color = "grey", width = 0.4, ylab = "Fold Enrichment", xlab = "", orientation = "horiz", sort.val = c("asc"))+theme(legend.position="none") 

print(p)

dev.off()