ED_Figure2_plots.R

suppressPackageStartupMessages(library(DESeq2))
suppressPackageStartupMessages(library(stringr))
suppressPackageStartupMessages(library(tidyverse))
suppressPackageStartupMessages(library(ggplot2))
suppressPackageStartupMessages(library(ggrepel))
suppressPackageStartupMessages(library(openxlsx))
suppressPackageStartupMessages(library(RColorBrewer))

## Input featureCounts file
fcount.file <- "Scarfo_HEC2023/BulkRNAseq_1/featureCounts_results_corrected.txt"
## Contrasts to perform
contr <- "ACE_pos:ACE_neg"
contr.list <- unlist(strsplit(contr, ","))
## Design Formula
formula.str <- "donor,condition"
design.str <- gsub(",", "+", formula.str)
num_cond <- str_count(formula.str, ",") + 1
design.formula <- as.formula(paste("~", design.str, sep = ""))
## Read featureCounts results
if (!file.exists(fcount.file)) {
  stop("Error: featureCounts file not found.")
} else {
  read.counts <- read.table(fcount.file, 
                            header = TRUE, 
                            check.names = FALSE) %>%
    column_to_rownames(var = "Geneid") %>%
    select(-c(1:5)) 
}
orig.names <- names(read.counts)
ds <- list()
cond <- list()
for (i in seq(orig.names[1:length(orig.names)])) {
  ds[i] <- unlist(strsplit(orig.names[i], ":"))[2]
  cond[i] <- unlist(strsplit(orig.names[i], ":"))[3]
}
ds <- unlist(ds)
cond <- unlist(cond)
## Samples - Conditions
sample_info <- as.data.frame(str_split_fixed(cond, ",", num_cond))
colnames(sample_info) <- str_split_fixed(formula.str, ",", num_cond)
rownames(sample_info) <- ds
colnames(read.counts) <- ds
sample_info$condition <- factor(sample_info$condition)
last_condition <- str_split_fixed(formula.str, ",", num_cond)[num_cond]
batch_cond <- str_split_fixed(formula.str, ",", num_cond)[num_cond - 1]
## thresholds
adj.pval.thr <- 0.05
logfc.thr <- 0
## DESeq2 
DESeq.ds <- DESeqDataSetFromMatrix(countData = read.counts,
                                   colData = sample_info,
                                   design = design.formula)
## Remove genes without any counts
DESeq.ds <- DESeq.ds[rowSums(counts(DESeq.ds)) > 0, ]
## RunDGE
DESeq.ds <- DESeq(DESeq.ds)
## obtain regularized log-transformed values
DESeq.rlog <- rlogTransformation(DESeq.ds, blind = TRUE)
assay(DESeq.rlog) <- limma::removeBatchEffect(assay(DESeq.rlog), DESeq.rlog[[batch_cond]])
ctrs <- unlist(strsplit(contr.list[1], ":"))
c1 <- ctrs[1]
c2 <- ctrs[2]
DGE.results <- results(DESeq.ds,
                       pAdjustMethod = "BH",
                       independentFiltering = TRUE,
                       contrast = c(last_condition, c1, c2),
                       alpha = adj.pval.thr)
# Change format
DGE.results.annot <- as.data.frame(DGE.results) %>% 
  dplyr::select(log2FoldChange, lfcSE, baseMean, pvalue, padj)
colnames(DGE.results.annot) <- c("logFC", "lfcSE", "baseMean", "PValue", "FDR")
## sort the results according to the adjusted p-value
DGE.results.sorted <- DGE.results.annot[order(DGE.results.annot$FDR), ]
## Subset for only significant genes
DGE.results.sig <- subset(DGE.results.sorted, 
                          FDR < adj.pval.thr & abs(logFC) > logfc.thr)
DGEgenes <- rownames(DGE.results.sig)


# ED_Fig2A ----------------------------------------------------------------

## surface genes
if(!file.exists("Scarfo_HEC2023/BulkRNAseq_1/table_S3_surfaceome.xlsx")){
  download.file("http://wlab.ethz.ch/surfaceome/table_S3_surfaceome.xlsx", 
                destfile = "Scarfo_HEC2023/BulkRNAseq_1/table_S3_surfaceome.xlsx")  
}
sg <- read.xlsx("Scarfo_HEC2023/BulkRNAseq_1/table_S3_surfaceome.xlsx", 
                sheet = "in silico surfaceome only",
                colNames = T,
                startRow = 2)
sig_sg <- DGE.results.sig[c(row.names(DGE.results.sig) %in% sg$UniProt.gene), ]
top10_sig_sg <- sig_sg[c(1:10),]
data <- data.frame(gene = row.names(DGE.results.sorted), 
                   pval = -log10(DGE.results.sorted$FDR), 
                   lfc = DGE.results.sorted$logFC)
data <- na.omit(data)
data <- mutate(data, color = case_when(data$lfc > logfc.thr & data$pval > -log10(adj.pval.thr) ~ "Overexpressed",data$lfc < -logfc.thr & data$pval > -log10(adj.pval.thr) ~ "Underexpressed",abs(data$lfc) < logfc.thr | data$pval < -log10(adj.pval.thr) ~ "NonSignificant"))
data$color <- factor(data$color, levels = c("Overexpressed", "Underexpressed", "NonSignificant"))
data <- data[order(data$pval, decreasing = TRUE),]
highl <- data[c(data$gene %in% row.names(top10_sig_sg)), ]
ggplot(data, aes(x = lfc, y = pval, colour = color)) +
  theme_bw() +
  theme(legend.position = "right",
        text=element_text(size=17*96/72),
        axis.text.x = element_text(size = 17*96/72, 
                                   color = "black"),
        axis.text.y = element_text(size = 17*96/72,
                                   color = "black"),
        axis.title.y = element_text(colour = "black", size = 19*96/72),
        axis.title.x = element_text(colour = "black", size = 19*96/72),
        legend.text = element_text(colour = "black", size = 19*96/72),
        legend.title = element_text(colour = "black", size = 19*96/72)) +
  xlab("log2 Fold Change") + 
  ylab(expression(-log[10]("adjusted p-value"))) +
  geom_hline(yintercept = -log10(adj.pval.thr), colour = "darkgray") +
  geom_point(size = 3, alpha = 0.8, na.rm = T) +
  scale_color_manual(name = "Expression",
                     values = c(Overexpressed = "red",
                                Underexpressed = "royalblue3",
                                NonSignificant = "darkgray")) +
  geom_label_repel(data = highl, 
                   aes(label = gene), 
                   show.legend = FALSE, 
                   size = 5)