diff --git a/bin/de_analysis.R b/bin/de_analysis.R index 8888b87..cedde4f 100755 --- a/bin/de_analysis.R +++ b/bin/de_analysis.R @@ -21,8 +21,8 @@ suppressMessages(library("edgeR")) # Some functions, including dmDSdata, converts '.' in sample IDs to '-'. # Make the output DF match the sample IDs in the sample sheet # start_col is the index of the first sample column in the data frame -rename_sample_columns <- function(df, sample_ids, start_col) { - colnames(df)[start_col:ncol(df)] <- sample_ids +rename_sample_columns <- function(df, alias, start_col) { + colnames(df)[start_col:ncol(df)] <- alias return(df) } @@ -37,13 +37,14 @@ if (!dir.exists(argv$merged_out_dir)){ cat("Loading counts, conditions and parameters.\n") cts <- as.matrix(read.csv(argv$all_counts, sep="\t", row.names="Reference", stringsAsFactors=FALSE, check.names=FALSE)) # Set up sample data frame: -#changed this to sample_id coldata <- read.csv(argv$sample_sheet, row.names="alias", sep=",", stringsAsFactors=TRUE, check.names=FALSE) +coldata$alias <- rownames(coldata) +#dmDSdata looks up sample_id in coldata, we need this to be sample alias +coldata$sample_id <- coldata$alias -coldata$sample_id <- rownames(coldata) # Reorder the input counts columns to match the sample sheet order. # This ensures we don't missassign column names, when renaming output DF columns. -cts <- cts[, coldata$sample_id, drop = FALSE] +cts <- cts[, coldata$alias, drop = FALSE] # check if control condition exists, sets as reference if(!"control" %in% coldata$condition) stop("sample_sheet.csv does not contain 'control' @@ -101,10 +102,11 @@ rownames(txdf) <- NULL # Create counts data frame: counts<-data.frame(gene_id=txdf$GENEID, feature_id=txdf$TXNAME, cts) -counts <- rename_sample_columns(counts, coldata$sample_id, 3) +counts <- rename_sample_columns(counts, coldata$alias, 3) # output unfiltered version of the counts table now we have paired transcripts with gene ids write.table(counts, file=file.path(argv$de_out_dir, "unfiltered_transcript_counts_with_genes.tsv"), sep="\t", row.names = FALSE, quote=FALSE) + cat("Filtering counts using DRIMSeq.\n") d <- dmDSdata(counts=counts, samples=coldata) @@ -122,13 +124,13 @@ suppressMessages(library("dplyr")) # Sum transcript counts into gene counts: cat("Sum transcript counts into gene counts.\n") trs_cts <- counts(d) -trs_cts <- rename_sample_columns(trs_cts, coldata$sample_id, 3) +trs_cts <- rename_sample_columns(trs_cts, coldata$alias, 3) write.table(trs_cts, file=file.path(argv$merged_out_dir, "filtered_transcript_counts_with_genes.tsv"), sep="\t", row.names = FALSE, quote=FALSE) gene_cts <- trs_cts_unfiltered %>% dplyr::select(c(1, 3:ncol(trs_cts))) %>% group_by(gene_id) %>% summarise_all(tibble::lst(sum)) %>% data.frame() rownames(gene_cts) <- gene_cts$gene_id gene_cts$gene_id <- NULL -gene_cts <- rename_sample_columns(gene_cts, coldata$sample_id, 1) +gene_cts <- rename_sample_columns(gene_cts, coldata$alias, 1) write.table(gene_cts, file=file.path(argv$merged_out_dir, "all_gene_counts.tsv"), sep="\t", quote=FALSE) # Output count per million of the gene counts using edgeR CPM @@ -137,7 +139,7 @@ cpm_gene_counts <- cpm(gene_cts) cpm_gene_counts <- cbind(var_name = rownames(cpm_gene_counts), cpm_gene_counts) rownames(cpm_gene_counts) <- NULL colnames(cpm_gene_counts)[1] <- "gene_id" -cpm_gene_counts <- rename_sample_columns(cpm_gene_counts, coldata$sample_id, 2) +cpm_gene_counts <- rename_sample_columns(cpm_gene_counts, coldata$alias, 2) write.table(cpm_gene_counts, file=file.path(argv$de_out_dir, "cpm_gene_counts.tsv"), sep="\t", quote=FALSE, row.names = FALSE) # Differential gene expression using edgeR: diff --git a/subworkflows/differential_expression.nf b/subworkflows/differential_expression.nf index 68c19ff..5266aab 100644 --- a/subworkflows/differential_expression.nf +++ b/subworkflows/differential_expression.nf @@ -78,6 +78,7 @@ process deAnalysis { path "de_analysis/results_dtu_stageR.tsv", emit: dtu_stageR path "de_analysis/results_dtu.pdf", emit: dtu_pdf path "de_analysis/cpm_gene_counts.tsv", emit: cpm + script: """ de_analysis.R \ --annotation annotation.gtf \ @@ -91,11 +92,13 @@ process deAnalysis { --merged_out_dir merged # Check that the original aliases in the input TSV have not been mangled by R's read.csv or other functions - head -1 all_counts.tsv | cut -f2- | tr '\t' '\n' > expected_colnames + # Sample column order should be in the same order as the input sample sheet + alias_col=\$(awk -v RS=',' '/alias/{print NR; exit}' "sample_sheet.csv") + cut -d',' -f3 sample_sheet.csv | tail -n +2 | paste -sd '\t' - | sed 's/\t*\$//' > expected_colnames - head -1 de_analysis/cpm_gene_counts.tsv | cut -f2- | tr '\t' '\n' > cpm_gene_counts_colnames - head -1 merged/all_gene_counts.tsv | tr '\t' '\n' > merged_counts_colnames - head -1 merged/filtered_transcript_counts_with_genes.tsv | cut -f3- | tr '\t' '\n' > merged_filtered_colnames + head -1 de_analysis/cpm_gene_counts.tsv | cut -f2- > cpm_gene_counts_colnames + head -1 merged/all_gene_counts.tsv > merged_counts_colnames + head -1 merged/filtered_transcript_counts_with_genes.tsv | cut -f3- > merged_filtered_colnames # Check for mismatches in sample column names for file in cpm_gene_counts_colnames merged_counts_colnames merged_filtered_colnames; do