Good morning.

I would like to plot an Enhanced Volcano using the results of my toptable. I am unable to plot a short list of genes of my own interest. Moreover, I created two cutoffs: one from adjusted p value and another from my beta variable (log2 fold change), and I found that my volcano is not right. These are my comands:

library(EnhancedVolcano)

NM53_WT_T_NM53_atf6_T<-NM53_WT_T_NM53_atf6_T %>% dplyr::distinct(ext_gene,.keep_all=T)

keyvals.colour <- ifelse(
  NM53_WT_T_NM53_atf6_T$b< -1.0 & NM53_WT_T_NM53_atf6_T$pval<10^-4, 'forestgreen',
  ifelse(NM53_WT_T_NM53_atf6_T$b > 1.0 & NM53_WT_T_NM53_atf6_T$pval<10^-4, 'red2',
         'grey30'))
keyvals.colour[is.na(keyvals.colour)] <- 'grey30'
names(keyvals.colour)[keyvals.colour == 'red2'] <- 'Upregulated genes'
names(keyvals.colour)[keyvals.colour == 'grey30'] <- 'NS'
names(keyvals.colour)[keyvals.colour == 'forestgreen'] <- 'Downregulated genes'

volcano.dir<-"F:/r_output/ES 2162/Volcano plots/"
setwd(volcano.dir)

s_genes<-c('Hspa5','Calr',"Pdia3","H2-D1")
pdf(paste0(volcano.dir,"_","NM53_WT_T_NM53_atf6_T","_",Sys.Date(),".pdf"),width = 9,height = 9,
    paper = "a4",onefile = T)
Volcano <- EnhancedVolcano(NM53_WT_T_NM53_atf6_T,
                      lab = NM53_WT_T_NM53_atf6_T$ext_gene,
                      x = 'b',
                      y = 'qval',
                      selectLab = s_genes,
                      xlim = c(-7,7),
                      ylim = c(0,9),
                      boxedLabels = TRUE,
                      xlab = "log2(Fold-change)",
                      ylab = "-log10(adjusted P-value)",
                      title = 'Volcano NM53_WT_T_NM53_atf6_T',
                      pCutoff = 10^-4,
                      FCcutoff = 1.0,
                      pointSize = 1.0,
                      labSize = 6.0,
                      labCol = "black",
                      labFace = "bold",
                      colCustom = keyvals.colour,
                      colAlpha = 4/5,
                      legendPosition = 'right',
                      legendLabSize = 10,
                      legendIconSize = 2.0,
                      drawConnectors = TRUE,
                      widthConnectors = 1.0,
                      typeConnectors = 'open',
                      endsConnectors = 'last',
                      colConnectors="black",
                      arrowheads = TRUE,
                      gridlines.major = TRUE,
                      gridlines.minor = FALSE,
                      border = 'partial',
                      borderWidth = 1.0,
                      borderColour = 'black')
Volcano
dev.off()

Could you help me?

Best regards