diff --git a/bu_isciii/templates/rnaseq/ANALYSIS/DATE_ANALYSIS01_RNASEQ/02-differential_expression/time_series_differential_expression.R b/bu_isciii/templates/rnaseq/ANALYSIS/DATE_ANALYSIS01_RNASEQ/02-differential_expression/time_series_differential_expression.R index d3358cd2..57d1725f 100644 --- a/bu_isciii/templates/rnaseq/ANALYSIS/DATE_ANALYSIS01_RNASEQ/02-differential_expression/time_series_differential_expression.R +++ b/bu_isciii/templates/rnaseq/ANALYSIS/DATE_ANALYSIS01_RNASEQ/02-differential_expression/time_series_differential_expression.R @@ -232,6 +232,49 @@ quality_plots <- function(norm_data){ dev.off() } +####TIME SERIES PLOTS######################### +time_series_plots <- function(gene, res, dds) { + plot_name <- paste(gene, "expression.pdf", sep = "_") + file_path <- paste("Time_series_plots", plot_name, sep = "/") + index = which(rownames(res) == gene, arr.ind = TRUE) + gene_name <- DE_results_merged[index,2] + fiss <- plotCounts(dds, index, + intgroup = c("time","condition"), returnData = TRUE) + fiss$time <- factor(fiss$time, levels = time_order) + p <- ggplot(fiss, aes(x = time, y = count, color = condition, group = condition)) + + geom_point() + + stat_summary(fun = mean, geom = "line") + + scale_y_log10() + + labs(title = paste("Expression evolution of gene: ", gene_name, sep = "")) + pdf(file=file_path) + print(p) + dev.off() +} + +####WALD TEST FOR TIME SERIES######################### + +wald_test <- function(dds, condition){ + condition_test <- results(dds, name=condition, test="Wald") + print(condition_test[which.min(condition_test$padj),]) +} + +####BETAS PLOT FOR TIME SERIES######################### + +betas_plot <- function(res, dds) { + betas <- coef(dds) + colnames(betas) + + topGenes <- head(order(res$padj),20) + mat <- betas[topGenes, -c(1,2)] + thr <- 3 + mat[mat < -thr] <- -thr + mat[mat > thr] <- thr + pdf(file="Time_series_plots/betas_pheatmap.pdf") + pheatmap(mat, breaks=seq(from=-thr, to=thr, length=101), + cluster_col=TRUE, main = "log2FC of top20 significant genes") + dev.off() +} + ################################################ ## WARNINGS ## ################################################ @@ -335,6 +378,23 @@ if (opt$quality_plots) { quality_plots(norm_data = norm_count) } +cat(blue("########################\nStarting with time series plots\n###############################\n")) + +top4 <- rownames(head(deseq2_results$dds_matrix[order(deseq2_results$results$padj),], 4)) + +dir.create("Time_series_plots",showWarnings = FALSE) + +for (gene in top4) { + time_series_plots(gene, res = deseq2_results$results, dds = deseq2_results$dds_matrix) +} + +all_conditions <- resultsNames(deseq2_results$dds_matrix) +for (condition in all_conditions) { + wald_test(dds = deseq2_results$dds_matrix, condition) +} + +betas_plot(res = deseq2_results$results, dds = deseq2_results$dds_matrix) + save.image() cat(blue("########################\nNumber of genes with padj < 0.05 and log2FC >= |2|:\n")) cat(blue(nrow(DE_results_merged_sig)))