## ----setup, include=FALSE, echo=F, warning= F, message=F---------------------- knitr::opts_chunk$set( message = FALSE, warning = FALSE, error = FALSE, tidy = FALSE, fig.align = "center", dpi = 150, cache = FALSE, progress = FALSE, quite = TRUE ) ## ---- echo=FALSE, results="hide", warning=FALSE,message=FALSE----------------- suppressPackageStartupMessages({ if("TCGAWorkflow" %in% installed.packages()[,1]) { library(TCGAWorkflow) } else { devtools::load_all(".") } }) ## ---- eval=FALSE, include=TRUE------------------------------------------------ # if (!"BiocManager" %in% rownames(installed.packages())) # install.packages("BiocManager") # BiocManager::install("TCGAWorkflow") # BiocManager::install("TCGAWorkflowData") ## ---- eval=TRUE, message=FALSE, warning=FALSE, include=TRUE------------------- library(TCGAWorkflowData) library(DT) ## ---- eval=TRUE, message=FALSE, warning=FALSE, include=FALSE------------------ library(TCGAbiolinks) ## ---- eval=FALSE, include=TRUE------------------------------------------------ # library(TCGAbiolinks) # # Obs: The data in the legacy database has been aligned to hg19 # query.met.gbm <- GDCquery( # project = "TCGA-GBM", # legacy = TRUE, # data.category = "DNA methylation", # platform = "Illumina Human Methylation 450", # barcode = c("TCGA-76-4926-01B-01D-1481-05", "TCGA-28-5211-01C-11D-1844-05") # ) # GDCdownload(query.met.gbm) # # met.gbm.450 <- GDCprepare( # query = query.met.gbm, # save = TRUE, # save.filename = "gbmDNAmet450k.rda", # summarizedExperiment = TRUE # ) # # query.met.lgg <- GDCquery( # project = "TCGA-LGG", # legacy = TRUE, # data.category = "DNA methylation", # platform = "Illumina Human Methylation 450", # barcode = c("TCGA-HT-7879-01A-11D-2399-05", "TCGA-HT-8113-01A-11D-2399-05") # ) # GDCdownload(query.met.lgg) # met.lgg.450 <- GDCprepare(query = query.met.lgg, # save = TRUE, # save.filename = "lggDNAmet450k.rda", # summarizedExperiment = TRUE) # met.gbm.lgg <- SummarizedExperiment::cbind(met.lgg.450, met.gbm.450) # # # query.exp.lgg <- GDCquery( # project = "TCGA-LGG", # legacy = TRUE, # data.category = "Gene expression", # data.type = "Gene expression quantification", # platform = "Illumina HiSeq", # file.type = "results", # sample.type = "Primary solid Tumor" # ) # GDCdownload(query.exp.lgg) # exp.lgg <- GDCprepare(query = query.exp.lgg, save = TRUE, save.filename = "lggExp.rda") # # query.exp.gbm <- GDCquery( # project = "TCGA-GBM", # legacy = TRUE, # data.category = "Gene expression", # data.type = "Gene expression quantification", # platform = "Illumina HiSeq", # file.type = "results", # sample.type = "Primary solid Tumor" # ) # GDCdownload(query.exp.gbm) # exp.gbm <- GDCprepare(query = query.exp.gbm, save = TRUE, save.filename = "gbmExp.rda") # exp.gbm.lgg <- SummarizedExperiment::cbind(exp.lgg, exp.gbm) ## ---- eval=FALSE, include=TRUE------------------------------------------------ # #----------------------------------------------------------------------------- # # Data.category: Copy number variation aligned to hg38 # #----------------------------------------------------------------------------- # query <- GDCquery( # project = "TCGA-ACC", # data.category = "Copy Number Variation", # data.type = "Copy Number Segment", # barcode = c( "TCGA-OR-A5KU-01A-11D-A29H-01", "TCGA-OR-A5JK-01A-11D-A29H-01")\ # ) # GDCdownload(query) # data <- GDCprepare(query) # # query <- GDCquery( # project = "TCGA-ACC", # data.category = "Copy Number Variation", # data.type = "Masked Copy Number Segment", # sample.type = c("Primary solid Tumor") # ) # see the barcodes with getResults(query)$cases # GDCdownload(query) # data <- GDCprepare(query) ## ---- eval=TRUE, include=TRUE------------------------------------------------- library(SummarizedExperiment) # Load object from TCGAWorkflowData package # THis object will be created in the further sections, data(GBMIllumina_HiSeq) # get expression matrix data <- assay(gbm.exp) datatable( data = data[1:10,], options = list(scrollX = TRUE, keys = TRUE, pageLength = 5), rownames = TRUE ) # get genes information genes.info <- rowRanges(gbm.exp) genes.info # get sample information sample.info <- colData(gbm.exp) datatable( data = as.data.frame(sample.info), options = list(scrollX = TRUE, keys = TRUE, pageLength = 5), rownames = FALSE ) ## ---- eval=TRUE, include=TRUE------------------------------------------------- # get indexed clinical patient data for GBM samples gbm_clin <- GDCquery_clinic(project = "TCGA-GBM", type = "Clinical") # get indexed clinical patient data for LGG samples lgg_clin <- GDCquery_clinic(project = "TCGA-LGG", type = "Clinical") # Bind the results, as the columns might not be the same, # we will will plyr rbind.fill, to have all columns from both files clinical <- plyr::rbind.fill(gbm_clin,lgg_clin) ## ----echo = TRUE, message = FALSE, warning = FALSE---------------------------- datatable(clinical[1:10,], options = list(scrollX = TRUE, keys = TRUE), rownames = FALSE) ## ----results = 'hide', echo=TRUE, message=FALSE, warning=FALSE---------------- # Fetch clinical data directly from the clinical XML files. # if barcode is not set, it will consider all samples. # We only set it to make the example faster query <- GDCquery( project = "TCGA-GBM", file.type = "xml", data.category = "Clinical", barcode = c("TCGA-08-0516","TCGA-02-0317") ) GDCdownload(query) clinical <- GDCprepare_clinic(query, clinical.info = "patient") ## ----echo = TRUE, message = FALSE, warning = FALSE---------------------------- datatable(clinical, options = list(scrollX = TRUE, keys = TRUE), rownames = FALSE) ## ----results = 'hide', echo=TRUE, message=FALSE, warning=FALSE---------------- clinical.drug <- GDCprepare_clinic(query, clinical.info = "drug") ## ----echo = TRUE, message = FALSE, warning = FALSE---------------------------- datatable(clinical.drug, options = list(scrollX = TRUE, keys = TRUE), rownames = FALSE) ## ----results = 'hide', echo=TRUE, message=FALSE, warning=FALSE---------------- clinical.radiation <- GDCprepare_clinic(query, clinical.info = "radiation") ## ----echo = TRUE, message = FALSE, warning = FALSE---------------------------- datatable(clinical.radiation, options = list(scrollX = TRUE, keys = TRUE), rownames = FALSE) ## ----results = 'hide', echo=TRUE, message=FALSE, warning=FALSE---------------- clinical.admin <- GDCprepare_clinic(query, clinical.info = "admin") ## ----echo = TRUE, message = FALSE, warning = FALSE---------------------------- datatable(clinical.admin, options = list(scrollX = TRUE, keys = TRUE), rownames = FALSE) ## ---- eval=FALSE, include=TRUE------------------------------------------------ # LGGmut <- GDCquery_Maf(tumor = "LGG", pipelines = "mutect2") ## ----echo = TRUE, message = FALSE, warning = FALSE---------------------------- data(mafMutect2LGGGBM) datatable(LGGmut[1:10,], options = list(scrollX = TRUE, keys = TRUE), rownames = FALSE) ## ---- eval=TRUE, include=TRUE------------------------------------------------- gbm.subtypes <- TCGAquery_subtype(tumor = "gbm") ## ----echo = TRUE, message = FALSE, warning = FALSE---------------------------- datatable(gbm.subtypes[1:10,], options = list(scrollX = TRUE, keys = TRUE), rownames = FALSE) ## ---- eval=FALSE, include=TRUE------------------------------------------------ # library(RTCGAToolbox) ## ---- eval=FALSE, include=TRUE------------------------------------------------ # # Get the last run dates # lastRunDate <- getFirehoseRunningDates()[1] # # # get DNA methylation data, RNAseq2 and clinical data for GBM # gbm.data <- getFirehoseData( # dataset = "GBM", # runDate = lastRunDate, # gistic2Date = getFirehoseAnalyzeDates(1), # Methylation = FALSE, # clinical = TRUE, # RNASeq2GeneNorm = FALSE, # Mutation = TRUE, # fileSizeLimit = 10000 # ) # # gbm.mut <- getData(gbm.data,"Mutation") # gbm.clin <- getData(gbm.data,"clinical") ## ---- eval=FALSE, message=FALSE, warning=FALSE, include=TRUE------------------ # # Download GISTIC results # lastanalyzedate <- getFirehoseAnalyzeDates(1) # gistic <- getFirehoseData("GBM",GISTIC = TRUE, gistic2Date = lastanalyzedate) # # # get GISTIC results # gistic.allbygene <- getData(gistic, type = "GISTIC", platform = "AllByGene") # gistic.thresholedbygene <- getData(gistic, type = "GISTIC", platform = "ThresholdedByGene") ## ---- eval=TRUE, message=FALSE, warning=FALSE, include=TRUE------------------- data(GBMGistic) gistic.allbygene %>% head() %>% gt::gt() gistic.thresholedbygene %>% head() %>% gt::gt() ## ----results='hide', eval=FALSE, message=FALSE, warning=FALSE, include=TRUE---- # library(TCGAbiolinks) # # Select common CN technology available for GBM and LGG # ############################# # ## CNV data pre-processing ## # ############################# # query.gbm.nocnv <- GDCquery( # project = "TCGA-GBM", # data.category = "Copy number variation", # legacy = TRUE, # file.type = "nocnv_hg19.seg", # sample.type = c("Primary solid Tumor") # ) # # to reduce time we will select only 20 samples # query.gbm.nocnv$results[[1]] <- query.gbm.nocnv$results[[1]][1:20,] # # GDCdownload(query.gbm.nocnv, files.per.chunk = 100) # # gbm.nocnv <- GDCprepare(query.gbm.nocnv, save = TRUE, save.filename = "GBMnocnvhg19.rda") ## ----results='hide', eval=FALSE, message=FALSE, warning=FALSE, include=TRUE---- # # -=-=-=-=-=-=-=-=-=-=-=-=-=-=-=--=-==--=--==---=-=-=-=-=-=-=-=-=-=-=-=-=-=-=--=-==--=--==-- # # Retrieve probes meta file from broadinstitute website for hg19 # # For hg38 analysis please take a look on: # # https://gdc.cancer.gov/about-data/data-harmonization-and-generation/gdc-reference-files # # File: SNP6 GRCh38 Liftover Probeset File for Copy Number Variation Analysis # # -=-=-=-=-=-=-=-=-=-=-=-=-=-=-=--=-==--=--==---=-=-=-=-=-=-=-=-=-=-=-=-=-=-=--=-==--=--==-- # gdac.root <- "ftp://ftp.broadinstitute.org/pub/GISTIC2.0/hg19_support/" # file <- paste0(gdac.root, "genome.info.6.0_hg19.na31_minus_frequent_nan_probes_sorted_2.1.txt") # if(!file.exists(basename(file))) downloader::download(file, basename(file)) # markersMatrix <- readr::read_tsv(basename(file), col_names = FALSE, col_types = "ccn", progress = FALSE) # save(markersMatrix, file = "markersMatrix.rda", compress = "xz") ## ----gaia, echo=TRUE, message=FALSE,warning=FALSE, include=TRUE--------------- cancer <- "GBM" message(paste0("Starting ", cancer)) # get objects created above data(GBMnocnvhg19) data(markersMatrix) head(cnvMatrix) head(markersMatrix) # Add label (0 for loss, 1 for gain) cnvMatrix <- cnvMatrix %>% dplyr::filter(abs(cnvMatrix$Segment_Mean) > 0.3) cnvMatrix$Aberration <- ifelse(cnvMatrix$Segment_Mean > 0.3, 1, 0) # Remove "Segment_Mean" and change col.names cnvMatrix$Segment_Mean <- NULL colnames(cnvMatrix) <- c("Sample.Name", "Chromosome", "Start", "End", "Num.of.Markers", "Aberration") # Substitute Chromosomes "X" and "Y" with "23" and "24" cnvMatrix$Chromosome[cnvMatrix$Chromosome == "X"] <- "23" cnvMatrix$Chromosome[cnvMatrix$Chromosome == "Y"] <- "24" cnvMatrix$Chromosome <- as.integer(cnvMatrix$Chromosome) cnvMatrix <- cnvMatrix %>% as.data.frame() # Recurrent CNV identification with GAIA colnames(markersMatrix) <- c("Probe.Name", "Chromosome", "Start") unique(markersMatrix$Chromosome) markersMatrix$Chromosome[markersMatrix$Chromosome == "X"] <- "23" markersMatrix$Chromosome[markersMatrix$Chromosome == "Y"] <- "24" markersMatrix$Chromosome <- as.integer(markersMatrix$Chromosome) markersMatrix <- markersMatrix %>% as.data.frame() markerID <- paste(markersMatrix$Chromosome,markersMatrix$Start, sep = ":") # Removed duplicates markersMatrix <- markersMatrix[!duplicated(markerID),] # Filter markersMatrix for common CNV markerID <- paste(markersMatrix$Chromosome,markersMatrix$Start, sep = ":") file <- "ftp://ftp.broadinstitute.org/pub/GISTIC2.0/hg19_support/CNV.hg19.bypos.111213.txt" if(!file.exists(basename(file))) downloader::download(file, basename(file)) commonCNV <- readr::read_tsv(basename(file), progress = FALSE) #data(CNV.hg19.bypos.111213) commonID <- paste(commonCNV$Chromosome,commonCNV$Start, sep = ":") markersMatrix_fil <- markersMatrix[!markerID %in% commonID,] library(gaia) set.seed(200) markers_obj <- load_markers( marker_matrix = markersMatrix_fil %>% as.data.frame() ) nbsamples <- length(unique(cnvMatrix$Sample.Name)) cnv_obj <- load_cnv( segmentation_matrix = cnvMatrix %>% as.data.frame(), markers_list = markers_obj, num_of_samples = nbsamples ) suppressWarnings({ results <- runGAIA( cnv_obj = cnv_obj, markers_obj = markers_obj, output_file_name = paste0("GAIA_",cancer,"_flt.txt"), aberrations = -1, # -1 to all aberrations chromosomes = 9, # -1 to all chromosomes approximation = TRUE, # Set to TRUE to speed up the time requirements num_iterations = 5000, # Reduced to speed up the time requirements threshold = 0.25) }) # Set q-value threshold # Use a smalled value for your analysis. We set this as high values # due to the small number of samples which did not reproduced # results with smaller q-values threshold <- 0.3 # Plot the results mode(results) <- "numeric" # our results are character we need to transform to numeric RecCNV <- results %>% as.data.frame() RecCNV$score <- 0 # replace 0 q-value to min non-zero minval <- format(min(RecCNV[RecCNV[,"q-value"] != 0,"q-value"]), scientific = FALSE) minval <- substring(minval,1, nchar(minval) - 1) %>% as.numeric RecCNV$`q-value`[RecCNV$`q-value` == 0] <- minval RecCNV$score <- -log10(RecCNV$`q-value`) RecCNV[RecCNV[,"q-value"] == as.numeric(minval),] gaiaCNVplot(RecCNV,threshold) save(results, RecCNV, threshold, file = paste0(cancer,"_CNV_results.rda")) ## ---- eval = TRUE, message=FALSE,warning=FALSE, include=FALSE----------------- library(GenomicRanges) ## ---- eval = FALSE, message=FALSE,warning=FALSE, include=TRUE----------------- # library(GenomicRanges) # # Get gene information from GENCODE using biomart # genes <- TCGAbiolinks:::get.GRCh.bioMart(genome = "hg19") # genes <- genes[genes$external_gene_name != "" & genes$chromosome_name %in% c(1:22,"X","Y"),] # genes[genes$chromosome_name == "X", "chromosome_name"] <- 23 # genes[genes$chromosome_name == "Y", "chromosome_name"] <- 24 # genes$chromosome_name <- sapply(genes$chromosome_name,as.integer) # genes <- genes[order(genes$start_position),] # genes <- genes[order(genes$chromosome_name),] # genes <- genes[,c("external_gene_name", "chromosome_name", "start_position","end_position")] # colnames(genes) <- c("GeneSymbol","Chr","Start","End") # genes_GR <- makeGRangesFromDataFrame(genes,keep.extra.columns = TRUE) # save(genes_GR,genes,file = "genes_GR.rda", compress = "xz") ## ---- echo=TRUE, message=FALSE,warning=FALSE, include=TRUE-------------------- ############################## ## Recurrent CNV annotation ## ############################## # Get gene information from GENCODE using biomart data(genes_GR) # downloaded in the previous step (available in TCGAWorkflowData) load(paste0(cancer,"_CNV_results.rda")) sCNV <- RecCNV[RecCNV[,"q-value"] <= threshold,c(1:4,6)] sCNV <- sCNV[order(sCNV$Chromosome,sCNV$`Region Start [bp]`),] colnames(sCNV) <- c("Chr","Aberration","Start","End","q-value") sCNV_GR <- makeGRangesFromDataFrame(sCNV,keep.extra.columns = TRUE) hits <- findOverlaps(genes_GR, sCNV_GR, type = "within") sCNV_ann <- cbind(sCNV[subjectHits(hits),],genes[queryHits(hits),]) AberrantRegion <- paste0(sCNV_ann[,1],":",sCNV_ann[,3],"-",sCNV_ann[,4]) GeneRegion <- paste0(sCNV_ann[,7],":",sCNV_ann[,8],"-",sCNV_ann[,9]) AmpDel_genes <- cbind(sCNV_ann[,c(6,2,5)],AberrantRegion,GeneRegion) AmpDel_genes[AmpDel_genes[,2] == 0,2] <- "Del" AmpDel_genes[AmpDel_genes[,2] == 1,2] <- "Amp" rownames(AmpDel_genes) <- NULL save(RecCNV, AmpDel_genes, file = paste0(cancer,"_CNV_results.rda")) ## ----results='asis', echo=FALSE, message=FALSE, warning=FALSE----------------- knitr::kable(head(AmpDel_genes), caption = "Chromosome 9 recurrent deleted genes in LGG") ## ----results='hide', echo=TRUE, message=FALSE, warning=FALSE, eval = FALSE---- # library(maftools) # # Download Mutation Annotation Format (MAF) files # LGGmut <- GDCquery_Maf(tumor = "LGG", pipelines = "mutect2") # GBMmut <- GDCquery_Maf(tumor = "GBM", pipelines = "mutect2") # # # Merge them # mut <- plyr::rbind.fill(LGGmut, GBMmut) # save(maf,file ="mafMutect2LGGGBM.rda", compress = "xz") ## ----results='hide', echo=TRUE, message=FALSE, warning=FALSE------------------ library(maftools) # recovering data from TCGAWorkflowData package. data(mafMutect2LGGGBM) # To prepare for maftools we will also include clinical data # For a mutant vs WT survival analysis # get indexed clinical patient data for GBM samples gbm_clin <- GDCquery_clinic(project = "TCGA-GBM", type = "Clinical") # get indexed clinical patient data for LGG samples lgg_clin <- GDCquery_clinic(project = "TCGA-LGG", type = "Clinical") # Bind the results, as the columns might not be the same, # we will will plyr rbind.fill, to have all columns from both files clinical <- plyr::rbind.fill(gbm_clin,lgg_clin) colnames(clinical)[1] <- "Tumor_Sample_Barcode" # we need to create a binary variable 1 is dead 0 is not dead plyr::count(clinical$vital_status) clinical$Overall_Survival_Status <- 1 # dead clinical$Overall_Survival_Status[which(clinical$vital_status != "Dead")] <- 0 # If patient is not dead we don't have days_to_death (NA) # we will set it as the last day we know the patient is still alive clinical$time <- clinical$days_to_death clinical$time[is.na(clinical$days_to_death)] <- clinical$days_to_last_follow_up[is.na(clinical$days_to_death)] # Create object to use in maftools maf <- read.maf(maf = mut, clinicalData = clinical, isTCGA = TRUE) ## ----echo=TRUE, message=FALSE, warning=FALSE,fig.width=10--------------------- plotmafSummary( maf = maf, rmOutlier = TRUE, addStat = 'median', dashboard = TRUE ) ## ----echo=TRUE, message=FALSE, warning=FALSE,fig.height=10,fig.width=15,eval=FALSE---- # oncoplot( # maf = maf, # top = 20, # legendFontSize = 8, # clinicalFeatures = c("tissue_or_organ_of_origin") # ) ## ----echo=TRUE, message=FALSE, warning=FALSE---------------------------------- plot <- mafSurvival( maf = maf, genes = "TP53", time = 'time', Status = 'Overall_Survival_Status', isTCGA = TRUE ) ## ----results='hide', eval = FALSE, echo=TRUE, message=FALSE,warning=FALSE----- # LGGmut <- GDCquery_Maf(tumor = "LGG", pipelines = "mutect2") ## ----results='hide', echo=TRUE, message=FALSE,warning=FALSE------------------- ############################################### ## Genomic aberration overview - Circos plot ## ############################################### # Retrieve curated mutations for selected cancer (e.g. "LGG") data(mafMutect2LGGGBM) # Select only potentially damaging mutations LGGmut %>% dplyr::filter(Variant_Classification %in% c( "Missense_Mutation", "Nonsense_Mutation", "Nonstop_Mutation", "Frame_Shift_Del", "Frame_Shift_Ins" ) ) # Select recurrent mutations (identified in at least two samples) mut.id <- paste0(LGGmut$Chromosome,":",LGGmut$Start_Position,"-",LGGmut$End_Position,"|",LGGmut$Reference_Allele) mut <- cbind(mut.id, LGGmut) # Prepare selected mutations data for circos plot s.mut <- mut[mut$mut.id %in% unique(mut.id[duplicated(mut.id)]),] s.mut <- s.mut[,c("Chromosome","Start_Position","End_Position","Variant_Classification","Hugo_Symbol")] s.mut <- unique(s.mut) typeNames <- unique(s.mut[,4]) type <- c(4:1) names(type) <- typeNames[1:4] Type <- type[s.mut[,4]] s.mut <- cbind(s.mut,Type) s.mut <- s.mut[,c(1:3,6,4,5)] # Load recurrent CNV data for selected cancer (e.g. "LGG") load("GBM_CNV_results.rda") # Prepare selected sample CNV data for circos plot s.cnv <- as.data.frame(RecCNV[RecCNV[,"q-value"] <= threshold,c(1:4,6)]) s.cnv <- s.cnv[,c(1,3,4,2)] s.cnv[s.cnv$Chromosome == 23,"Chromosome"] <- "X" s.cnv[s.cnv$Chromosome == 24,"Chromosome"] <- "Y" Chromosome <- paste0("chr",s.cnv[,1]) s.cnv <- cbind(Chromosome, s.cnv[,-1]) s.cnv[,1] <- as.character(s.cnv[,1]) s.cnv[,4] <- as.character(s.cnv[,4]) s.cnv <- cbind(s.cnv,CNV=1) colnames(s.cnv) <- c("Chromosome","Start_position","End_position","Aberration_Kind","CNV") library(circlize) # Draw genomic circos plot par(mar=c(1,1,1,1), cex=1) circos.initializeWithIdeogram() # Add CNV results colors <- c("forestgreen","firebrick") names(colors) <- c(0,1) circos.genomicTrackPlotRegion( s.cnv, ylim = c(0,1.2), panel.fun = function(region, value, ...) { circos.genomicRect(region, value, ytop.column = 2, ybottom = 0, col = colors[value[[1]]], border="white") cell.xlim = get.cell.meta.data("cell.xlim") circos.lines(cell.xlim, c(0, 0), lty = 2, col = "#00000040") }) # Add mutation results colors <- c("blue","green","red","gold") names(colors) <- typeNames[1:4] circos.genomicTrackPlotRegion( s.mut, ylim = c(1.2,4.2), panel.fun = function(region, value, ...) { circos.genomicPoints(region, value, cex = 0.8, pch = 16, col = colors[value[[2]]], ...) }) circos.clear() legend( -0.2, 0.2, bty = "n", y.intersp = 1, c("Amp", "Del"), pch = 15, col = c("firebrick", "forestgreen"), title = "CNVs", text.font = 1, cex = 0.4, title.adj = 0 ) legend( -0.2, 0, bty = "n", y.intersp = 1, names(colors), pch = 16, col = colors, title = "Mutations", text.font = 1, cex = 0.4, title.adj = 0 ) ## ----results='asis', echo=TRUE, message=FALSE,warning=FALSE------------------- par(mar=c(1,1,1,1),cex=1.5) circos.par( "start.degree" = 90, canvas.xlim = c(0, 1), canvas.ylim = c(0, 1), gap.degree = 270, cell.padding = c(0, 0, 0, 0), track.margin = c(0.005, 0.005) ) circos.initializeWithIdeogram(chromosome.index = "chr17") circos.par(cell.padding = c(0, 0, 0, 0)) # Add CNV results colors <- c("forestgreen","firebrick") names(colors) <- c(0,1) circos.genomicTrackPlotRegion( data = s.cnv, ylim = c(0,1.2), panel.fun = function(region, value, ...) { circos.genomicRect(region, value, ytop.column = 2, ybottom = 0, col = colors[value[[1]]], border="white") cell.xlim = get.cell.meta.data("cell.xlim") circos.lines(cell.xlim, c(0, 0), lty = 2, col = "#00000040") } ) # Add mutation results representing single genes genes.mut <- paste0(s.mut$Hugo_Symbol,"-",s.mut$Type) s.mutt <- cbind(s.mut,genes.mut) n.mut <- table(genes.mut) idx <- !duplicated(s.mutt$genes.mut) s.mutt <- s.mutt[idx,] s.mutt <- cbind(s.mutt,num=n.mut[s.mutt$genes.mut]) genes.num <- paste0(s.mutt$Hugo_Symbol," (",s.mutt$num.Freq,")") s.mutt <- cbind(s.mutt[,-c(6:8)],genes.num) s.mutt[,6] <- as.character(s.mutt[,6]) s.mutt[,4] <- s.mutt[,4]/2 s.mutt$num.Freq <- NULL colors <- c("blue","green","red","gold") names(colors) <- typeNames[1:4] circos.genomicTrackPlotRegion( data = s.mutt, ylim = c(0.3,2.2), track.height = 0.05, panel.fun = function(region, value, ...) { circos.genomicPoints(region, value, cex = 0.4, pch = 16, col = colors[value[[2]]], ...) } ) circos.genomicTrackPlotRegion(s.mutt, ylim = c(0, 1), track.height = 0.1, bg.border = NA) i_track = get.cell.meta.data("track.index") circos.genomicTrackPlotRegion( data = s.mutt, ylim = c(0,1), panel.fun = function(region, value, ...) { circos.genomicText( region, value, y = 1, labels.column = 3, col = colors[value[[2]]], facing = "clockwise", adj = c(1, 0.5), posTransform = posTransform.text, cex = 0.4, niceFacing = TRUE) }, track.height = 0.1, bg.border = NA ) circos.genomicPosTransformLines( data = s.mutt, posTransform = function(region, value) posTransform.text(region, y = 1, labels = value[[3]], cex = 0.4, track.index = i_track+1), direction = "inside", track.index = i_track ) circos.clear() legend( x = 0.25, y = 0.2, bty = "n", y.intersp = 1, c("Amp","Del"), pch = 15, col = c("firebrick","forestgreen"), title = "CNVs", text.font = 1, cex = 0.4, title.adj = 0 ) legend( x = 0, y = 0.2, bty = "n", y.intersp = 1, names(colors), pch = 16, col = colors, title = "Mutations", text.font = 1, cex = 0.4, title.adj = 0 ) ## ---- eval=FALSE, include=TRUE------------------------------------------------ # query <- GDCquery( # project = "TCGA-GBM", # data.category = "Gene expression", # data.type = "Gene expression quantification", # platform = "Illumina HiSeq", # file.type = "results", # sample.type = c("Primary solid Tumor"), # legacy = TRUE # ) # # We will use only 20 samples to make the example faster # query$results[[1]] <- query$results[[1]][1:20,] # GDCdownload(query) # gbm.exp <- GDCprepare( # query = query, # save = TRUE, # summarizedExperiment = TRUE, # save.filename = "GBMIllumina_HiSeq.rda" # ) # # query <- GDCquery( # project = "TCGA-LGG", # data.category = "Gene expression", # data.type = "Gene expression quantification", # platform = "Illumina HiSeq", # file.type = "results", # sample.type = c("Primary solid Tumor"), # legacy = TRUE # ) # # We will use only 20 samples to make the example faster # query$results[[1]] <- query$results[[1]][1:20,] # GDCdownload(query) # lgg.exp <- GDCprepare( # query = query, # save = TRUE, # summarizedExperiment = TRUE, # save.filename = "LGGIllumina_HiSeq.rda" # ) ## ----results='asis', echo=TRUE, message=FALSE,warning=FALSE------------------- data("LGGIllumina_HiSeq") data("GBMIllumina_HiSeq") dataPrep_LGG <- TCGAanalyze_Preprocessing( object = lgg.exp, cor.cut = 0.6, datatype = "raw_count", filename = "LGG_IlluminaHiSeq_RNASeqV2.png" ) dataPrep_GBM <- TCGAanalyze_Preprocessing( object = gbm.exp, cor.cut = 0.6, datatype = "raw_count", filename = "GBM_IlluminaHiSeq_RNASeqV2.png" ) dataNorm <- TCGAanalyze_Normalization( tabDF = cbind(dataPrep_LGG, dataPrep_GBM), geneInfo = TCGAbiolinks::geneInfo, method = "gcContent" ) #18323 672 dataFilt <- TCGAanalyze_Filtering( tabDF = dataNorm, method = "quantile", qnt.cut = 0.25 ) # 13742 672 save(dataFilt, file = paste0("LGG_GBM_Norm_IlluminaHiSeq.rda")) dataFiltLGG <- subset( dataFilt, select = substr(colnames(dataFilt),1,12) %in% lgg_clin$bcr_patient_barcode ) dataFiltGBM <- subset( dataFilt, select = substr(colnames(dataFilt),1,12) %in% gbm_clin$bcr_patient_barcode ) dataDEGs <- TCGAanalyze_DEA( mat1 = dataFiltLGG, mat2 = dataFiltGBM, Cond1type = "LGG", Cond2type = "GBM", fdr.cut = 0.01 , logFC.cut = 1, method = "glmLRT" ) ## ----results='asis', echo=TRUE, message=FALSE,warning=FALSE------------------- # Number of differentially expressed genes (DEG) nrow(dataDEGs) ## ----results='hide', echo=TRUE, message=FALSE,warning=FALSE,fig.height=10, fig.width=10---- #------------------- 4.2 EA: enrichment analysis -------------------- ansEA <- TCGAanalyze_EAcomplete( TFname = "DEA genes LGG Vs GBM", RegulonList = rownames(dataDEGs) ) TCGAvisualize_EAbarplot( tf = rownames(ansEA$ResBP), filename = NULL, GOBPTab = ansEA$ResBP, nRGTab = rownames(dataDEGs), nBar = 20) ## ----results='hide', echo=TRUE, message=FALSE,warning=FALSE,fig.height=10, fig.width=10---- TCGAvisualize_EAbarplot( tf = rownames(ansEA$ResBP), filename = NULL, GOCCTab = ansEA$ResCC, nRGTab = rownames(dataDEGs), nBar = 20 ) ## ----results='hide', echo=TRUE, message=FALSE,warning=FALSE,fig.height=10, fig.width=10---- TCGAvisualize_EAbarplot( tf = rownames(ansEA$ResBP), filename = NULL, GOMFTab = ansEA$ResMF, nRGTab = rownames(dataDEGs), nBar = 20 ) ## ----results='hide', echo=TRUE, message=FALSE,warning=FALSE,fig.height=12, fig.width=15---- TCGAvisualize_EAbarplot( tf = rownames(ansEA$ResBP), filename = NULL, PathTab = ansEA$ResPat, nRGTab = rownames(dataDEGs), nBar = 20 ) ## ----results='asis', echo=TRUE, message=FALSE,warning=FALSE------------------- library(SummarizedExperiment) GenelistComplete <- rownames(assay(lgg.exp,1)) # DEGs TopTable dataDEGsFiltLevel <- TCGAanalyze_LevelTab( FC_FDR_table_mRNA = dataDEGs, typeCond1 = "LGG", typeCond2 = "GBM", TableCond1 = dataFilt[,colnames(dataFiltLGG)], TableCond2 = dataFilt[,colnames(dataFiltGBM)] ) dataDEGsFiltLevel$GeneID <- 0 library(clusterProfiler) # Converting Gene symbol to geneID eg = as.data.frame( bitr(dataDEGsFiltLevel$mRNA, fromType = "SYMBOL", toType = "ENTREZID", OrgDb = "org.Hs.eg.db") ) eg <- eg[!duplicated(eg$SYMBOL),] dataDEGsFiltLevel <- dataDEGsFiltLevel[dataDEGsFiltLevel$mRNA %in% eg$SYMBOL,] dataDEGsFiltLevel <- dataDEGsFiltLevel[order(dataDEGsFiltLevel$mRNA,decreasing=FALSE),] eg <- eg[order(eg$SYMBOL,decreasing=FALSE),] # table(eg$SYMBOL == dataDEGsFiltLevel$mRNA) should be TRUE all(eg$SYMBOL == dataDEGsFiltLevel$mRNA) dataDEGsFiltLevel$GeneID <- eg$ENTREZID dataDEGsFiltLevel_sub <- subset(dataDEGsFiltLevel, select = c("GeneID", "logFC")) genelistDEGs <- as.numeric(dataDEGsFiltLevel_sub$logFC) names(genelistDEGs) <- dataDEGsFiltLevel_sub$GeneID library(pathview) # pathway.id: hsa05214 is the glioma pathway # limit: sets the limit for gene expression legend and color hsa05214 <- pathview::pathview( gene.data = genelistDEGs, pathway.id = "hsa05214", species = "hsa", limit = list(gene = as.integer(max(abs(genelistDEGs)))) ) ## ---- eval=FALSE, include=TRUE------------------------------------------------ # ### read biogrid info (available in TCGAWorkflowData as "biogrid") # ### Check last version in https://thebiogrid.org/download.php # file <- "http://thebiogrid.org/downloads/archives/Release%20Archive/BIOGRID-3.4.146/BIOGRID-ALL-3.4.146.tab2.zip" # if(!file.exists(gsub("zip","txt",basename(file)))){ # downloader::download(file,basename(file)) # unzip(basename(file),junkpaths =TRUE) # } # # tmp.biogrid <- read.csv(gsub("zip","txt",basename(file)), header=TRUE, sep="\t", stringsAsFactors=FALSE) ## ---- eval=TRUE, include=TRUE------------------------------------------------- ### plot details (colors & symbols) mycols <- c('#e41a1c','#377eb8','#4daf4a','#984ea3','#ff7f00','#ffff33','#a65628') ### load network inference libraries library(minet) library(c3net) ### deferentially identified genes using TCGAbiolinks # we will use only a subset (first 50 genes) of it to make the example faster names.genes.de <- rownames(dataDEGs)[1:30] data(biogrid) net.biogrid.de <- getAdjacencyBiogrid(tmp.biogrid, names.genes.de) mydata <- dataFiltLGG[names.genes.de, ] ### infer networks t.mydata <- t(mydata) net.aracne <- minet(t.mydata, method = "aracne") net.mrnet <- minet(t.mydata) net.clr <- minet(t.mydata, method = "clr") net.c3net <- c3net(mydata) ### validate compared to biogrid network tmp.val <- list( validate(net.aracne, net.biogrid.de), validate(net.mrnet, net.biogrid.de), validate(net.clr, net.biogrid.de), validate(net.c3net, net.biogrid.de) ) ### plot roc and compute auc for the different networks dev1 <- show.roc(tmp.val[[1]],cex=0.3,col=mycols[1],type="l") res.auc <- auc.roc(tmp.val[[1]]) for(count in 2:length(tmp.val)){ show.roc(tmp.val[[count]],device=dev1,cex=0.3,col=mycols[count],type="l") res.auc <- c(res.auc, auc.roc(tmp.val[[count]])) } legend("bottomright", legend=paste(c("aracne","mrnet","clr","c3net"), signif(res.auc,4), sep=": "), col=mycols[1:length(tmp.val)],lty=1, bty="n" ) # Please, uncomment this line to produce the pdf files. # dev.copy2pdf(width=8,height=8,device = dev1, file = paste0("roc_biogrid_",cancertype,".pdf")) ## ---- eval=FALSE, include=TRUE------------------------------------------------ # #---------------------------- # # Obtaining DNA methylation # #---------------------------- # # Samples # lgg.samples <- matchedMetExp("TCGA-LGG", n = 10) # gbm.samples <- matchedMetExp("TCGA-GBM", n = 10) # samples <- c(lgg.samples,gbm.samples) # # #----------------------------------- # # 1 - Methylation # # ---------------------------------- # # For methylation it is quicker in this case to download the tar.gz file # # and get the samples we want instead of downloading files by files # query <- GDCquery( # project = c("TCGA-LGG","TCGA-GBM"), # data.category = "DNA methylation", # platform = "Illumina Human Methylation 450", # legacy = TRUE, # barcode = samples # ) # GDCdownload(query) # met <- GDCprepare(query, save = FALSE) # # # We will use only chr9 to make the example faster # met <- subset(met,subset = as.character(seqnames(met)) %in% c("chr9")) # # This data is avaliable in the package (object elmerExample) ## ----echo=TRUE, message=FALSE,warning=FALSE, fig.height=8,fig.width=8--------- data(elmerExample) #---------------------------- # Mean methylation #---------------------------- # Plot a barplot for the groups in the disease column in the # summarizedExperiment object # remove probes with NA (similar to na.omit) met <- met[rowSums(is.na(assay(met))) == 0,] df <- data.frame( "Sample.mean" = colMeans(assay(met), na.rm = TRUE), "groups" = met$project_id ) library(ggpubr) ggpubr::ggboxplot( data = df, y = "Sample.mean", x = "groups", color = "groups", add = "jitter", ylab = expression(paste("Mean DNA methylation (", beta, "-values)")), xlab = "" ) + stat_compare_means() ## ----results='hide', echo=TRUE, message=FALSE,warning=FALSE------------------- #------- Searching for differentially methylated CpG sites ---------- dmc <- TCGAanalyze_DMC( data = met, groupCol = "project_id", # a column in the colData matrix group1 = "TCGA-GBM", # a type of the disease type column group2 = "TCGA-LGG", # a type of the disease column p.cut = 0.05, diffmean.cut = 0.15, save = FALSE, legend = "State", plot.filename = "LGG_GBM_metvolcano.png", cores = 1 # if set to 1 there will be a progress bar ) ## ----results='hide', echo=TRUE, message=FALSE,warning=FALSE------------------- #-------------------------- # DNA Methylation heatmap #------------------------- library(ComplexHeatmap) clinical <- plyr::rbind.fill(gbm_clin,lgg_clin) # get the probes that are Hypermethylated or Hypomethylated # met is the same object of the section 'DNA methylation analysis' status.col <- "status" probes <- rownames(dmc)[grep("hypo|hyper",dmc$status,ignore.case = TRUE)] sig.met <- met[probes,] # top annotation, which sampples are LGG and GBM # We will add clinical data as annotation of the samples # we will sort the clinical data to have the same order of the DNA methylation matrix clinical.order <- clinical[match(substr(colnames(sig.met),1,12),clinical$bcr_patient_barcode),] ta <- HeatmapAnnotation( df = clinical.order[, c("disease", "gender", "vital_status", "race")], col = list( disease = c("LGG" = "grey", "GBM" = "black"), gender = c("male" = "blue", "female" = "pink") ) ) # row annotation: add the status for LGG in relation to GBM # For exmaple: status.gbm.lgg Hypomethyated means that the # mean DNA methylation of probes for lgg are hypomethylated # compared to GBM ones. ra = rowAnnotation( df = dmc[probes, status.col], col = list( "status.TCGA.GBM.TCGA.LGG" = c("Hypomethylated" = "orange", "Hypermethylated" = "darkgreen") ), width = unit(1, "cm") ) heatmap <- Heatmap( matrix = assay(sig.met), name = "DNA methylation", col = matlab::jet.colors(200), show_row_names = FALSE, cluster_rows = TRUE, cluster_columns = FALSE, show_column_names = FALSE, bottom_annotation = ta, column_title = "DNA Methylation" ) # Save to pdf png("heatmap.png",width = 600, height = 400) draw(heatmap, annotation_legend_side = "bottom") dev.off() ## ----results='asis', echo=TRUE, message=FALSE,warning=FALSE------------------- library(rGADEM) library(BSgenome.Hsapiens.UCSC.hg19) library(motifStack) library(SummarizedExperiment) library(dplyr) probes <- rowRanges(met)[rownames(dmc)[grep("hypo|hyper",dmc$status,ignore.case = TRUE)],] # Get hypo/hyper methylated probes and make a 200bp window # surrounding each probe. sequence <- GRanges( seqnames = as.character(seqnames(probes)), IRanges( start = ranges(probes) %>% as.data.frame() %>% dplyr::pull("start") - 100, end = ranges(probes) %>% as.data.frame() %>% dplyr::pull("end") + 100), strand = "*" ) #look for motifs gadem <- GADEM(sequence, verbose = FALSE, genome = Hsapiens) # How many motifs were found? nMotifs(gadem) # get the number of occurrences nOccurrences(gadem) # view all sequences consensus consensus(gadem) # Print motif pwm <- getPWM(gadem) pfm <- new("pfm",mat = pwm[[1]],name = "Novel Site 1") plotMotifLogo(pfm) # Number of instances of motif 1? length(gadem@motifList[[1]]@alignList) ## ----table4, echo=FALSE, message=FALSE, warnings=FALSE, results='asis'-------- tabl <- " | Histone marks | Role | |:----------------------------------------------:|:-------------------------------------------------------------------------------------------------------:| | Histone H3 lysine 4 trimethylation (H3K4me3) | Promoter regions [@heintzman2007distinct,@bernstein2005genomic] | | Histone H3 lysine 4 monomethylation (H3K4me1) | Enhancer regions [@heintzman2007distinct] | | Histone H3 lysine 36 trimethylation (H3K36me3) | Transcribed regions | | Histone H3 lysine 27 trimethylation (H3K27me3) | Polycomb repression [@bonasio2010molecular] | | Histone H3 lysine 9 trimethylation (H3K9me3) | Heterochromatin regions [@peters2003partitioning] | | Histone H3 acetylated at lysine 27 (H3K27ac) | Increase activation of genomic elements [@heintzman2009histone,@rada2011unique,@creyghton2010histone] | | Histone H3 lysine 9 acetylation (H3K9ac) | Transcriptional activation [@nishida2006histone] | " cat(tabl) ## ----table5, echo=FALSE, message=FALSE, warnings=FALSE, results='asis'-------- tabl <- " | File | Description | |:----------------------------------:|:----------------------------------------------------------------------:| | fc.signal.bigwig | Bigwig File containing fold enrichment signal tracks | | pval.signal.bigwig | Bigwig File containing -log10(p-value) signal tracks | | hotspot.fdr0.01.broad.bed.gz | Broad domains on enrichment for DNase-seq for consolidated epigenomes | | hotspot.broad.bed.gz | Broad domains on enrichment for DNase-seq for consolidated epigenomes | | broadPeak.gz | Broad ChIP-seq peaks for consolidated epigenomes | | gappedPeak.gz | Gapped ChIP-seq peaks for consolidated epigenomes | | narrowPeak.gz | Narrow ChIP-seq peaks for consolidated epigenomes | | hotspot.fdr0.01.peaks.bed.gz | Narrow DNasePeaks for consolidated epigenomes | | hotspot.all.peaks.bed.gz | Narrow DNasePeaks for consolidated epigenomes | | .macs2.narrowPeak.gz | Narrow DNasePeaks for consolidated epigenomes | | coreMarks_mnemonics.bed.gz | 15 state chromatin segmentations | | mCRF_FractionalMethylation.bigwig | MeDIP/MRE(mCRF) fractional methylation calls | | RRBS_FractionalMethylation.bigwig | RRBS fractional methylation calls | | WGBS_FractionalMethylation.bigwig | Whole genome bisulphite fractional methylation calls | " cat(tabl) ## ----results='hide', eval=TRUE, echo=TRUE, message=FALSE,warning=FALSE-------- library(ChIPseeker) library(pbapply) library(ggplot2) ## ----results='hide', eval=FALSE, echo=TRUE, message=FALSE,warning=FALSE------- # #------------------ Working with ChipSeq data --------------- # # Step 1: download histone marks for a brain and non-brain samples. # #------------------------------------------------------------ # # loading annotation hub database # library(AnnotationHub) # ah = AnnotationHub() # # # Searching for brain consolidated epigenomes in the roadmap database # bpChipEpi_brain <- query(ah , c("EpigenomeRoadMap", "narrowPeak", "chip", "consolidated","brain","E068")) # # Get chip-seq data # histone.marks <- pblapply(names(bpChipEpi_brain), function(x) {ah[[x]]}) # names(histone.marks) <- names(bpChipEpi_brain) # # OBS: histone.marks is available in TCGAWorkflowData package ## ----results='hide', echo=TRUE, message=FALSE,warning=FALSE------------------- data(histoneMarks) # Create a GR object based on the hypo/hypermethylated probes. probes <- keepStandardChromosomes(rowRanges(met)[rownames(dmc)[dmc$status %in% c("Hypermethylated in TCGA-GBM", "Hypomethylated in TCGA-GBM")],]) # Defining a window of 3kbp - 3kbp_probe_3kbp ranges(probes) <- IRanges(start = c(start(ranges(probes)) - 3000), end = c(start(ranges(probes)) + 3000)) ### Profile of ChIP peaks binding to TSS regions # First of all, to calculate the profile of ChIP peaks binding to TSS regions, we should # prepare the TSS regions, which are defined as the flanking sequence of the TSS sites. # Then align the peaks that are mapping to these regions and generate the tagMatrix. tagMatrixList <- pbapply::pblapply(histone.marks, function(x) { getTagMatrix(keepStandardChromosomes(x), windows = probes, weightCol = "score") }) # change names retrieved with the following command: basename(bpChipEpi_brain$title) names(tagMatrixList) <- c("H3K4me1","H3K4me3", "H3K9ac", "H3K9me3", "H3K27ac", "H3K27me3", "H3K36me3") ## ---- eval=FALSE, include=TRUE------------------------------------------------ # tagHeatmap(tagMatrixList, xlim=c(-3000, 3000),color = NULL) ## ----results='asis', echo=FALSE, fig.width = 7, message=FALSE,warning=FALSE---- tagHeatmap(tagMatrixList, xlim=c(-3000, 3000),color = NULL) ## ---- eval=FALSE, include=TRUE------------------------------------------------ # p <- plotAvgProf(tagMatrixList, xlim = c(-3000,3000), xlab = "Genomic Region (5'->3', centered on CpG)") # # We are centreing in the CpG instead of the TSS. So we'll change the labels manually # p <- p + scale_x_continuous(breaks=c(-3000,-1500,0,1500,3000),labels=c(-3000,-1500,"CpG",1500,3000)) # library(ggthemes) # p + theme_few() + scale_colour_few(name="Histone marks") + guides(colour = guide_legend(override.aes = list(size=4))) ## ----results='asis', echo=FALSE, message=FALSE,warning=FALSE------------------ p <- plotAvgProf(tagMatrixList, xlim = c(-3000,3000), xlab = "Genomic Region (5'->3', centered on CpG)") # We are centreing in the CpG instead of the TSS. So we'll change the labels manually if (requireNamespace("ggplot2", quietly = TRUE)) p <- p + ggplot2::scale_x_continuous(breaks=c(-3000,-1500,0,1500,3000), labels=c(-3000,-1500,"CpG",1500,3000)) if (requireNamespace("ggthemes", quietly = TRUE)) p + ggthemes::theme_few() + ggthemes::scale_colour_few(name="Histone marks") + ggplot2::guides(colour = guide_legend(override.aes = list(size=4))) ## ---- eval=FALSE, include=TRUE------------------------------------------------ # #----------- 8.3 Identification of Regulatory Enhancers ------- # library(TCGAbiolinks) # # Samples: primary solid tumor w/ DNA methylation and gene expression # lgg.samples <- matchedMetExp("TCGA-LGG", n = 10) # gbm.samples <- matchedMetExp("TCGA-GBM", n = 10) # samples <- c(lgg.samples,gbm.samples) # # #----------------------------------- # # 1 - Methylation # # ---------------------------------- # query.met <- GDCquery( # project = c("TCGA-LGG","TCGA-GBM"), # data.category = "DNA methylation", # platform = "Illumina Human Methylation 450", # legacy = TRUE, # barcode = samples # ) # GDCdownload(query.met) # met <- GDCprepare(query.met, save = FALSE) # met <- subset(met,subset = as.character(GenomicRanges::seqnames(met)) %in% c("chr9")) # # #----------------------------------- # # 2 - Expression # # ---------------------------------- # query.exp <- GDCquery( # project = c("TCGA-LGG","TCGA-GBM"), # data.category = "Gene expression", # data.type = "Gene expression quantification", # platform = "Illumina HiSeq", # file.type = "results", # legacy = TRUE, # barcode = samples # ) # GDCdownload(query.exp) # exp <- GDCprepare(query.exp, save = FALSE) # save(exp, met, gbm.samples, lgg.samples, file = "elmer.example.rda", compress = "xz") ## ---- message = FALSE,warning = FALSE----------------------------------------- library(ELMER) library(MultiAssayExperiment) library(GenomicRanges) distal.probes <- get.feature.probe(genome = "hg19", met.platform = "450K") # Recover the data created in the last step data(elmerExample) rownames(exp) <- values(exp)$ensembl_gene_id mae <- createMAE( exp = assay(exp), met = met, save = TRUE, linearize.exp = TRUE, save.filename = "mae.rda", filter.probes = distal.probes, met.platform = "450K", genome = "hg19", TCGA = TRUE ) mae ## ---- warning=FALSE, results="hide"------------------------------------------- cores <- 1 # you can use more cores if you want group.col <- "project_id" group1 <- "TCGA-GBM" group2 <- "TCGA-LGG" # Available directions are hypo and hyper, we will use only hypo # due to speed constraint direction <- "hypo" dir.out <- paste0("elmer/",direction) dir.create(dir.out, showWarnings = FALSE, recursive = TRUE) ## ---- warning=FALSE, results="hide"------------------------------------------- #-------------------------------------- # STEP 3: Analysis | #-------------------------------------- # Step 3.1: Get diff methylated probes | #-------------------------------------- message("Get diff methylated probes") Sig.probes <- get.diff.meth( data = mae, group.col = group.col, group1 = group1, group2 = group2, minSubgroupFrac = 1.0, # Use all samples sig.dif = 0.2, # defualt is 0.3 diff.dir = direction, # Search for hypomethylated probes in group 1 cores = cores, dir.out = dir.out, pvalue = 0.1 ) ## ---- warning=FALSE----------------------------------------------------------- datatable( data = Sig.probes[1:10,], options = list(scrollX = TRUE, keys = TRUE, pageLength = 5), rownames = TRUE ) ## ---- warning=FALSE, results="hide"------------------------------------------- #------------------------------------------------------------- # Step 3.2: Identify significant probe-gene pairs | #------------------------------------------------------------- # Collect nearby 20 genes for Sig.probes message("Get nearby genes") nearGenes <- GetNearGenes( data = mae, numFlankingGenes = 4, # default is 20 genes probes = Sig.probes$probe ) ## ----------------------------------------------------------------------------- length(Sig.probes$probe) dim(nearGenes) head(nearGenes) ## ---- warning=FALSE, results="hide"------------------------------------------- message("Get anti correlated probes-genes") pair <- get.pair( data = mae, group.col = group.col, group1 = group1, group2 = group2, nearGenes = nearGenes, mode = "supervised", minSubgroupFrac = 1, # % of samples to use in to create groups U/M raw.pvalue = 0.1, # defualt is 0.001 Pe = 0.5, # Please set to 0.001 to get significant results filter.probes = TRUE, # See preAssociationProbeFiltering function filter.percentage = 0.05, save = FALSE, # Create CVS file filter.portion = 0.3, dir.out = dir.out, diff.dir = direction, cores = cores, label = direction ) ## ---- warning=FALSE----------------------------------------------------------- datatable( pair[1:10,], options = list(scrollX = TRUE, keys = TRUE, pageLength = 5), rownames = TRUE ) ## ---- warning=FALSE, results="hide"------------------------------------------- #------------------------------------------------------------- # Step 3.3: Motif enrichment analysis on the selected probes | #------------------------------------------------------------- enriched.motif <- get.enriched.motif( data = mae, probes = pair$Probe, dir.out = dir.out, label = direction, pvalue = 1, # default is FDR < 0.05 min.incidence = 10, lower.OR = 1.1 ) # One of the output from the previous function is a file with the motif, OR and Number of probes # It will be used for plotting purposes motif.enrichment <- read.csv(paste0(dir.out,"/getMotif.",direction, ".motif.enrichment.csv")) ## ---- warning=FALSE----------------------------------------------------------- head(enriched.motif[names(enriched.motif)[1]]) ## probes in the given set that have the first motif. motif.enrichment %>% head %>% gt::gt() ## ---- warning=FALSE, results="hide"------------------------------------------- #------------------------------------------------------------- # Step 3.4: Identifying regulatory TFs | #------------------------------------------------------------- TF <- get.TFs( data = mae, group.col = group.col, group1 = group1, group2 = group2, mode = "supervised", enriched.motif = enriched.motif, dir.out = dir.out, cores = cores, save.plots = FALSE, diff.dir = direction, label = direction ) # One of the output from the previous function is a file with the raking of TF, # for each motif. It will be used for plotting purposes TF.meth.cor <- get(load(paste0(dir.out,"/getTF.",direction,".TFs.with.motif.pvalue.rda"))) ## ---- warning=FALSE----------------------------------------------------------- datatable(TF, options = list(scrollX = TRUE, keys = TRUE, pageLength = 5), rownames = FALSE) datatable(TF.meth.cor[1:10,1:6], options = list(scrollX = TRUE, keys = TRUE, pageLength = 5), rownames = TRUE) ## ---- echo=TRUE, message=FALSE,eval = FALSE, warnings=FALSE, results='asis',fig.height=6, fig.width=10---- # heatmapPairs( # data = mae, # group.col = group.col, # group1 = group1, # group2 = group2, # annotation.col = c("gender"), # pairs = pair, # filename = NULL # ) ## ---- echo=TRUE, message=FALSE, warnings=FALSE, results='asis',fig.height=7,fig.width=15---- motif.enrichment.plot( motif.enrichment = motif.enrichment, save = FALSE, significant = list(lowerOR = 1.1) ) # Filter motifs in the plot lowerOR > 1.3 ## ---- eval=FALSE, include=TRUE------------------------------------------------ # grid:TF.rank.plot(motif.pvalue=TF.meth.cor, motif=TF$motif[1], save=FALSE) ## ----results='asis', echo=FALSE, message=FALSE,warning=FALSE, fig.align='center',fig.height=8,fig.width=8---- gg <- TF.rank.plot(motif.pvalue = TF.meth.cor, motif=TF$motif[1], save=FALSE) grid::grid.draw(gg[[1]]) ## ----results='asis', echo=TRUE, message=FALSE, warning=FALSE------------------ png("TF.png",width = 800, height = 400) scatter.plot( data = mae, category = group.col, save = FALSE, lm_line = TRUE, byTF = list( TF = unlist(stringr::str_split(TF[1,"top_5percent_TFs"],";"))[1:4], probe = enriched.motif[[TF$motif[1]]] ) ) dev.off() ## ----------------------------------------------------------------------------- pander::pander(sessionInfo(), compact = FALSE)