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Conti_GE_2025_RNAseq_plots.R

+library(clusterProfiler)
+library(ggplot2)
+library(dplyr)
+library(gtools)
+library(RColorBrewer)
+
+# Working Directory
+wdir <- "~/conti_ge_2025_RNAseq"
+
+# Results 24h: paired_stranded24h
+wdir24 <- "paired_stranded24h"
+contr24 <- "GE_24h-RNP_NEG_24h"
+sen24 <- paste(wdir, wdir24, paste0(contr24, "-gsea-senescence_signatures.txt"), sep = "/")
+sen24_res <- read.table(sen24, sep = "\t", header = T)
+sasp24 <- paste(wdir, wdir24, paste0(contr24, "-gsea-sasp.txt"), sep = "/")
+sasp24_res <- read.table(sasp24, sep = "\t", header = T)
+sen24_res <- rbind(sen24_res, sasp24_res)
+sen24_terms <- c("SenMayo", "Purcell", "Pribluda_SENESCENCE_INFLAMMATORY_GENES", "Sencan", "SASP_SCHLEICH")
+sen24_res <- subset(sen24_res, ID %in% sen24_terms)
+sen24_res$List <- "Senescence"
+
+hmark24 <- paste(wdir, wdir24, paste0(contr24, "-gsea-h.all.v7.2.symbols.txt"), sep = "/")
+hmark24_res <- read.table(hmark24, sep = "\t", header = T)
+hmark24_res$List <- "Hallmark"
+res24 <- as.data.frame(rbind(sen24_res, hmark24_res) %>% select(ID, NES, pvalue, p.adjust, List))
+res24$Time <- "Day 1"
+res24$Contr <- contr24
+head(res24)
+
+# Results 96h: unstrand_96h
+wdir96 <- "unstrand_96h"
+contr96 <- "GE_96h-RNP_NEG_96h"
+sen96 <- paste(wdir, wdir96, paste0(contr96, "-gsea-senescence_signatures.txt"), sep = "/")
+sen96_res <- read.table(sen96, sep = "\t", header = T)
+sasp96 <- paste(wdir, wdir96, paste0(contr96, "-gsea-sasp.txt"), sep = "/")
+sasp96_res <- read.table(sasp96, sep = "\t", header = T)
+sen96_res <- rbind(sen96_res, sasp96_res)
+sen96_terms <- c("SenMayo", "Purcell", "Pribluda_SENESCENCE_INFLAMMATORY_GENES", "Sencan", "SASP_SCHLEICH")
+sen96_res <- subset(sen96_res, ID %in% sen96_terms)
+sen96_res$List <- "Senescence"
+
+hmark96 <- paste(wdir, wdir96, paste0(contr96, "-gsea-h.all.v7.2.symbols.txt"), sep = "/")
+hmark96_res <- read.table(hmark96, sep = "\t", header = T)
+hmark96_res$List <- "Hallmark"
+res96 <- as.data.frame(rbind(sen96_res, hmark96_res) %>% select(ID, NES, pvalue, p.adjust, List))
+res96$Time <- "Day 4"
+res96$Contr <- contr96
+
+res <- rbind(res24, res96)
+res$ID <- gsub("HALLMARK_", "", res$ID)
+sel_terms <- res %>% filter(p.adjust < 0.05 | List == "Senescence") %>% select(ID) %>% unique()
+res <- subset(res, ID %in% sel_terms$ID)
+ord_terms <- res %>% group_by(ID) %>% summarise(NES = mean(NES)) %>% arrange(NES)
+res$ID <- factor(res$ID, levels = ord_terms$ID)
+res$Sig <- stars.pval(p.value = res$pvalue)
+
+p <- ggplot(data = res, mapping = aes(x = Time, y = ID, fill = NES, label = Sig)) +
+    theme_bw(base_size = 14) +
+    theme(panel.spacing = unit(0, "lines"),
+          plot.title = element_text(hjust=0, vjust = -1.5),
+          panel.border = element_rect(color = "white"),
+          strip.background.y = element_rect(fill= "white", color = "white"),
+          strip.text.x = element_text(size = 14),
+          strip.text.y = element_blank(),
+          axis.text.x = element_blank(),
+          axis.ticks.x = element_blank(),
+          strip.background.x = element_rect(fill= "white", color = "white")) +
+    geom_tile(color = "white", lwd = .5, linetype = 1, width = 1.2) +
+    ylab("") +
+    xlab("") +
+    ggtitle("HS/AAV6 vs. Ctrl") +
+    scale_fill_gradient2(low = "darkblue", high = "darkred", mid = "grey") +
+    geom_text(size = 5, color = "white") +
+    facet_grid(List~Time, scales = "free", space = "free_y", switch="x")
+
+ggsave(filename = paste(wdir, "HM_GEvsRNPNEG_SenHallmark.png", sep = "/"),
+       plot = p, width = 7, height = 6)
+
+#########################################################
+# GSEA 2
+hallmark_terms <- c("KRAS_SIGNALING_UP","ESTROGEN_RESPONSE_LATE","MYOGENESIS","EPITHELIAL_MESENCHYMAL_TRANSITION","ANGIOGENESIS",
+                    "COAGULATION","HEDGEHOG_SIGNALING","IL6_JAK_STAT3_SIGNALING","TNFA_SIGNALING_VIA_NFKB","IL2_STAT5_SIGNALING",
+                    "INTERFERON_ALPHA_RESPONSE","INTERFERON_GAMMA_RESPONSE","MYC_TARGETS_V2","MITOTIC_SPINDLE","DNA_REPAIR",
+                    "UNFOLDED_PROTEIN_RESPONSE","G2M_CHECKPOINT","E2F_TARGETS","MTORC1_SIGNALING","MYC_TARGETS_V1","APICAL_JUNCTION",
+                    "UV_RESPONSE_UP","APOPTOSIS","REACTIVE_OXYGEN_SPECIES_PATHWAY","ESTROGEN_RESPONSE_EARLY","P53_PATHWAY",
+                    "ALLOGRAFT_REJECTION","XENOBIOTIC_METABOLISM","PROTEIN_SECRETION","OXIDATIVE_PHOSPHORYLATION","HEME_METABOLISM",
+                    "UV_RESPONSE_DN","INFLAMMATORY_RESPONSE","COMPLEMENT","BILE_ACID_METABOLISM","KRAS_SIGNALING_DN")
+
+# Results 24h: paired_stranded24h
+wdir24 <- "paired_stranded24h"
+contr24 <- "GE_ANAK_24h-GE_24h"
+hallm24_file <- paste(wdir, wdir24, paste0(contr24, "-gsea-h.all.v7.2.symbols.txt"), sep = "/")
+hallm24 <- read.table(hallm24_file, sep = "\t", header = T)
+hallm24$ID <- gsub("HALLMARK_", "", hallm24$ID)
+hallm24 <- subset(hallm24, ID %in% hallmark_terms)
+hallm24$List <- "Hallmark"
+head(hallm24)
+res24 <- hallm24 %>% select(ID, NES, pvalue, p.adjust, List)
+rownames(res24) <- seq(1, nrow(res24))
+res24$Time <- "Day 1"
+
+# Results 96h: unstrand_96h
+wdir96 <- "unstrand_96h"
+contr96 <- "GE_ANAK_96h-GE_96h"
+hallm96_file <- paste(wdir, wdir96, paste0(contr96, "-gsea-h.all.v7.2.symbols.txt"), sep = "/")
+hallm96 <- read.table(hallm96_file, sep = "\t", header = T)
+hallm96$ID <- gsub("HALLMARK_", "", hallm96$ID)
+hallm96 <- subset(hallm96, ID %in% hallmark_terms)
+hallm96$List <- "Hallmark"
+head(hallm96)
+res96 <- hallm96 %>% select(ID, NES, pvalue, p.adjust, List)
+rownames(res96) <- seq(1, nrow(res96))
+res96$Time <- "Day 4"
+
+res <- rbind(res24, res96)
+ord_terms <- res %>% group_by(ID) %>% summarise(NES = mean(NES)) %>% arrange(desc(NES))
+res$ID <- factor(res$ID, levels = ord_terms$ID)
+
+p <- ggplot(data = res, mapping = aes(x = ID, y = Time, fill = NES)) +
+    theme_bw(base_size = 14) +
+    theme(plot.title = element_text(hjust=.5),
+          panel.border = element_rect(color = "white"),
+          panel.grid = element_line(color = "white"),
+          axis.text.x = element_text(angle = 90, hjust = 1, vjust = 0.5),
+          strip.background.y = element_rect(fill= "white", color = "white"),
+          strip.text = element_blank(),
+          strip.background.x = element_rect(fill= "white", color = "white")) +
+    geom_tile(color = "white") +
+    ylab("") +
+    xlab("") +
+    ggtitle("HS/AAV6+ANAK vs. HS/AAV6") +
+    scale_fill_gradient2(low = "darkblue", high = "darkred", mid = "white", limits = c(-1.6,1.6)) +
+    scale_y_discrete(position = "right") +
+    facet_grid(.~List, scales = "free_x", space = "free_x")
+
+ggsave(filename = paste(wdir, "HM_GEANAKvsGE_Hallmark.png", sep = "/"),
+       plot = p, width = 12, height = 6)