## ----echo=FALSE---------------------------------------------------------------

  suppressWarnings(library(knitr))

  opts_chunk$set(tidy = FALSE, message = FALSE, warning = FALSE)


## ----getPackage, eval=FALSE---------------------------------------------------
# 
#   if (!requireNamespace('BiocManager', quietly = TRUE))
#     install.packages('BiocManager')
# 
#   BiocManager::install('EnhancedVolcano')
# 

## ----getPackageDevel, eval=FALSE----------------------------------------------
# 
#   devtools::install_github('kevinblighe/EnhancedVolcano')
# 

## ----Load, message=FALSE------------------------------------------------------

  library(EnhancedVolcano)


## -----------------------------------------------------------------------------

  library(airway)
  library(magrittr)

  data('airway')
  airway$dex %<>% relevel('untrt')


## -----------------------------------------------------------------------------

  ens <- rownames(airway)

  library(org.Hs.eg.db)
  symbols <- mapIds(org.Hs.eg.db, keys = ens,
    column = c('SYMBOL'), keytype = 'ENSEMBL')
  symbols <- symbols[!is.na(symbols)]
  symbols <- symbols[match(rownames(airway), names(symbols))]
  rownames(airway) <- symbols
  keep <- !is.na(rownames(airway))
  airway <- airway[keep,]


## -----------------------------------------------------------------------------

  library('DESeq2')

  dds <- DESeqDataSet(airway, design = ~ cell + dex)
  dds <- DESeq(dds, betaPrior=FALSE)
  res <- results(dds,
    contrast = c('dex','trt','untrt'))
  res <- lfcShrink(dds,
    contrast = c('dex','trt','untrt'), res=res, type = 'normal')


## ----ex1, fig.height = 8.5, fig.width = 7, fig.cap = "Plot the most basic volcano plot."----

  EnhancedVolcano(res,
    lab = rownames(res),
    x = 'log2FoldChange',
    y = 'pvalue')


## ----ex2, fig.height = 8.5, fig.width = 7, fig.cap = "Modify cut-offs for log2FC and P value; specify title; adjust point and label size."----

  EnhancedVolcano(res,
    lab = rownames(res),
    x = 'log2FoldChange',
    y = 'pvalue',
    title = 'N061011 versus N61311',
    pCutoff = 10e-32,
    FCcutoff = 0.5,
    pointSize = 3.0,
    labSize = 6.0)


## ----ex3, fig.height = 8.5, fig.width = 7, fig.cap = "Adjust colour and alpha for point shading."----

  EnhancedVolcano(res,
    lab = rownames(res),
    x = 'log2FoldChange',
    y = 'pvalue',
    title = 'N061011 versus N61311',
    pCutoff = 10e-16,
    FCcutoff = 1.5,
    pointSize = 3.0,
    labSize = 6.0,
    col=c('black', 'black', 'black', 'red3'),
    colAlpha = 1)


## ----ex4a, eval = FALSE-------------------------------------------------------
# 
#  EnhancedVolcano(res,
#     lab = rownames(res),
#     x = 'log2FoldChange',
#     y = 'pvalue',
#     title = 'N061011 versus N61311',
#     pCutoff = 10e-16,
#     FCcutoff = 1.5,
#     pointSize = 4.0,
#     labSize = 6.0,
#     shape = 8,
#     colAlpha = 1)
# 

## ----ex4b, fig.height = 8.5, fig.width = 7, fig.cap = "Adjust shape of plotted points."----

  EnhancedVolcano(res,
    lab = rownames(res),
    x = 'log2FoldChange',
    y = 'pvalue',
    title = 'N061011 versus N61311',
    pCutoff = 10e-16,
    FCcutoff = 1.5,
    pointSize = 3.0,
    labSize = 6.0,
    shape = c(1, 4, 23, 25),
    colAlpha = 1)


## ----ex5, fig.height = 8.5, fig.width = 7, fig.cap = "Adjust cut-off lines and add extra threshold lines."----

  EnhancedVolcano(res,
    lab = rownames(res),
    x = 'log2FoldChange',
    y = 'pvalue',
    xlim = c(-6, 6),
    title = 'N061011 versus N61311',
    pCutoff = 10e-12,
    FCcutoff = 1.5,
    pointSize = 3.0,
    labSize = 6.0,
    colAlpha = 1,
    cutoffLineType = 'blank',
    cutoffLineCol = 'black',
    cutoffLineWidth = 0.8,
    hline = c(10e-20,
      10e-20 * 10e-30,
      10e-20 * 10e-60,
      10e-20 * 10e-90),
    hlineCol = c('pink', 'hotpink', 'purple', 'black'),
    hlineType = c('solid', 'longdash', 'dotdash', 'dotted'),
    hlineWidth = c(1.0, 1.5, 2.0, 2.5),
    gridlines.major = FALSE,
    gridlines.minor = FALSE)


## ----ex6, fig.height = 8.5, fig.width = 10, fig.cap = "Adjust legend position, size, and text."----

  EnhancedVolcano(res,
    lab = rownames(res),
    x = 'log2FoldChange',
    y = 'pvalue',
    pCutoff = 10e-12,
    FCcutoff = 1.5,
    cutoffLineType = 'twodash',
    cutoffLineWidth = 0.8,
    pointSize = 4.0,
    labSize = 6.0,
    colAlpha = 1,
    legendLabels=c('Not sig.','Log (base 2) FC','p-value',
      'p-value & Log (base 2) FC'),
    legendPosition = 'right',
    legendLabSize = 16,
    legendIconSize = 5.0)


## ----eval = FALSE-------------------------------------------------------------
# 
# legendPosition = 'none'
# 

## ----ex7, fig.height = 8.5, fig.width = 10, fig.cap = "Fit more labels by adding connectors."----

  EnhancedVolcano(res,
    lab = rownames(res),
    x = 'log2FoldChange',
    y = 'pvalue',
    xlab = bquote(~Log[2]~ 'fold change'),
    pCutoff = 10e-32,
    FCcutoff = 2.0,
    pointSize = 4.0,
    labSize = 6.0,
    colAlpha = 1,
    legendPosition = 'right',
    legendLabSize = 12,
    legendIconSize = 4.0,
    drawConnectors = TRUE,
    widthConnectors = 0.75)


## ----ex8, eval = FALSE--------------------------------------------------------
# 
#   EnhancedVolcano(res,
#     lab = rownames(res),
#     x = 'log2FoldChange',
#     y = 'pvalue',
#     selectLab = c('TMEM176B','ADH1A'),
#     xlab = bquote(~Log[2]~ 'fold change'),
#     pCutoff = 10e-14,
#     FCcutoff = 2.0,
#     pointSize = 4.0,
#     labSize = 6.0,
#     shape = c(4, 35, 17, 18),
#     colAlpha = 1,
#     legendPosition = 'right',
#     legendLabSize = 14,
#     legendIconSize = 5.0)
# 

## ----ex9, fig.height = 8.5, fig.width = 10, fig.cap = "Draw labels in boxes."----

  EnhancedVolcano(res,
    lab = rownames(res),
    x = 'log2FoldChange',
    y = 'pvalue',
    selectLab = c('VCAM1','KCTD12','ADAM12',
      'CXCL12','CACNB2','SPARCL1','DUSP1','SAMHD1','MAOA'),
    xlab = bquote(~Log[2]~ 'fold change'),
    pCutoff = 10e-14,
    FCcutoff = 2.0,
    pointSize = 4.0,
    labSize = 6.0,
    labCol = 'black',
    labFace = 'bold',
    boxedLabels = TRUE,
    colAlpha = 4/5,
    legendPosition = 'right',
    legendLabSize = 14,
    legendIconSize = 4.0,
    drawConnectors = TRUE,
    widthConnectors = 1.0,
    colConnectors = 'black')


## ----ex10, fig.height = 7, fig.width = 10, fig.cap = "Italicise labels and flip volcano on it's side."----

  lab_italics <- paste0("italic('", rownames(res), "')")
  selectLab_italics = paste0(
    "italic('",
    c('VCAM1','KCTD12','ADAM12', 'CXCL12','CACNB2','SPARCL1','DUSP1','SAMHD1','MAOA'),
    "')")

  EnhancedVolcano(res,
    lab = lab_italics,
    x = 'log2FoldChange',
    y = 'pvalue',
    selectLab = selectLab_italics,
    xlab = bquote(~Log[2]~ 'fold change'),
    pCutoff = 10e-14,
    FCcutoff = 1.0,
    pointSize = 3.0,
    labSize = 6.0,
    labCol = 'black',
    labFace = 'bold',
    boxedLabels = TRUE,
    parseLabels = TRUE,
    col = c('black', 'pink', 'purple', 'red3'),
    colAlpha = 4/5,
    legendPosition = 'bottom',
    legendLabSize = 14,
    legendIconSize = 4.0,
    drawConnectors = TRUE,
    widthConnectors = 1.0,
    colConnectors = 'black') + coord_flip()


## ----eval = TRUE--------------------------------------------------------------

  # create custom key-value pairs for 'high', 'low', 'mid' expression by fold-change
  # this can be achieved with nested ifelse statements
  keyvals <- ifelse(
    res$log2FoldChange < -2.5, 'royalblue',
      ifelse(res$log2FoldChange > 2.5, 'gold',
        'black'))
  keyvals[is.na(keyvals)] <- 'black'
  names(keyvals)[keyvals == 'gold'] <- 'high'
  names(keyvals)[keyvals == 'black'] <- 'mid'
  names(keyvals)[keyvals == 'royalblue'] <- 'low'


## ----ex11, fig.height = 8.5, fig.width = 10, fig.cap = "Over-ride colouring scheme with custom key-value pairs."----

  EnhancedVolcano(res,
    lab = rownames(res),
    x = 'log2FoldChange',
    y = 'pvalue',
    selectLab = rownames(res)[which(names(keyvals) %in% c('high', 'low'))],
    xlab = bquote(~Log[2]~ 'fold change'),
    title = 'Custom colour over-ride',
    pCutoff = 10e-14,
    FCcutoff = 1.0,
    pointSize = 3.5,
    labSize = 4.5,
    shape = c(6, 4, 2, 11),
    colCustom = keyvals,
    colAlpha = 1,
    legendPosition = 'left',
    legendLabSize = 15,
    legendIconSize = 5.0,
    drawConnectors = TRUE,
    widthConnectors = 1.0,
    colConnectors = 'black',
    arrowheads = FALSE,
    gridlines.major = TRUE,
    gridlines.minor = FALSE,
    border = 'partial',
    borderWidth = 1.5,
    borderColour = 'black')


## ----eval = TRUE--------------------------------------------------------------

  # define different cell-types that will be shaded
  celltype1 <- c('VCAM1','KCTD12','ADAM12','CXCL12')
  celltype2 <- c('CACNB2','SPARCL1','DUSP1','SAMHD1','MAOA')

  # create custom key-value pairs for different cell-types
  # this can be achieved with nested ifelse statements
  keyvals.shape <- ifelse(
    rownames(res) %in% celltype1, 17,
      ifelse(rownames(res) %in% celltype2, 64,
        3))
  keyvals.shape[is.na(keyvals.shape)] <- 3
  names(keyvals.shape)[keyvals.shape == 3] <- 'PBMC'
  names(keyvals.shape)[keyvals.shape == 17] <- 'Cell-type 1'
  names(keyvals.shape)[keyvals.shape == 64] <- 'Cell-type 2'


## ----ex12, fig.height = 8.5, fig.width = 17, fig.cap = "Over-ride colour and/or shape scheme with custom key-value pairs."----

  p1 <- EnhancedVolcano(res,
    lab = rownames(res),
    x = 'log2FoldChange',
    y = 'pvalue',
    selectLab = rownames(res)[which(names(keyvals) %in% c('high', 'low'))],
    xlab = bquote(~Log[2]~ 'fold change'),
    title = 'Custom shape over-ride',
    pCutoff = 10e-14,
    FCcutoff = 1.0,
    pointSize = 4.5,
    labSize = 4.5,
    shapeCustom = keyvals.shape,
    colCustom = NULL,
    colAlpha = 1,
    legendLabSize = 15,
    legendPosition = 'left',
    legendIconSize = 5.0,
    drawConnectors = TRUE,
    widthConnectors = 0.5,
    colConnectors = 'grey50',
    gridlines.major = TRUE,
    gridlines.minor = FALSE,
    border = 'partial',
    borderWidth = 1.5,
    borderColour = 'black')

  # create custom key-value pairs for 'high', 'low', 'mid' expression by fold-change
  # this can be achieved with nested ifelse statements
  keyvals.colour <- ifelse(
    res$log2FoldChange < -2.5, 'royalblue',
      ifelse(res$log2FoldChange > 2.5, 'gold',
        'black'))
  keyvals.colour[is.na(keyvals.colour)] <- 'black'
  names(keyvals.colour)[keyvals.colour == 'gold'] <- 'high'
  names(keyvals.colour)[keyvals.colour == 'black'] <- 'mid'
  names(keyvals.colour)[keyvals.colour == 'royalblue'] <- 'low'

  p2 <- EnhancedVolcano(res,
    lab = rownames(res),
    x = 'log2FoldChange',
    y = 'pvalue',
    selectLab = rownames(res)[which(names(keyvals) %in% c('High', 'Low'))],
    xlab = bquote(~Log[2]~ 'fold change'),
    title = 'Custom shape & colour over-ride',
    pCutoff = 10e-14,
    FCcutoff = 1.0,
    pointSize = 5.5,
    labSize = 0.0,
    shapeCustom = keyvals.shape,
    colCustom = keyvals.colour,
    colAlpha = 1,
    legendPosition = 'right',
    legendLabSize = 15,
    legendIconSize = 5.0,
    drawConnectors = TRUE,
    widthConnectors = 0.5,
    colConnectors = 'grey50',
    gridlines.major = TRUE,
    gridlines.minor = FALSE,
    border = 'full',
    borderWidth = 1.0,
    borderColour = 'black')

  library(gridExtra)
  library(grid)
  grid.arrange(p1, p2,
    ncol=2,
    top = textGrob('EnhancedVolcano',
      just = c('center'),
      gp = gpar(fontsize = 32)))


## ----eval = TRUE--------------------------------------------------------------

  # define different cell-types that will be shaded
  celltype1 <- c('VCAM1','CXCL12')
  celltype2 <- c('SORT1', 'KLF15')


## ----ex13, fig.height = 8.5, fig.width = 10, fig.cap = "Shade certain variables."----

  EnhancedVolcano(res,
    lab = rownames(res),
    x = 'log2FoldChange',
    y = 'pvalue',
    selectLab = c(celltype1, celltype2),
    xlab = bquote(~Log[2]~ 'fold change'),
    title = 'Shading cell-type 1|2',
    pCutoff = 10e-14,
    FCcutoff = 1.0,
    pointSize = 8.0,
    labSize = 6.0,
    labCol = 'black',
    labFace = 'bold',
    boxedLabels = TRUE,
    shape = 42,
    colCustom = keyvals,
    colAlpha = 1,
    legendPosition = 'right',
    legendLabSize = 20,
    legendIconSize = 20.0,
    # encircle
      encircle = celltype1,
      encircleCol = 'black',
      encircleSize = 2.5,
      encircleFill = 'pink',
      encircleAlpha = 1/2,
    # shade
      shade = celltype2,
      shadeAlpha = 1/2,
      shadeFill = 'skyblue',
      shadeSize = 1,
      shadeBins = 5,
    drawConnectors = TRUE,
    widthConnectors = 2.0,
    gridlines.major = TRUE,
    gridlines.minor = FALSE,
    border = 'full',
    borderWidth = 5,
    borderColour = 'black')


## ----ex14, fig.height = 9, fig.width = 12, fig.cap = "Highlighting key variabvles via custom point sizes."----

  library("pasilla")
  pasCts <- system.file("extdata", "pasilla_gene_counts.tsv",
    package="pasilla", mustWork=TRUE)
  pasAnno <- system.file("extdata", "pasilla_sample_annotation.csv",
    package="pasilla", mustWork=TRUE)
  cts <- as.matrix(read.csv(pasCts,sep="\t",row.names="gene_id"))
  coldata <- read.csv(pasAnno, row.names=1)
  coldata <- coldata[,c("condition","type")]
  rownames(coldata) <- sub("fb", "", rownames(coldata))
  cts <- cts[, rownames(coldata)]
  library("DESeq2")
  dds <- DESeqDataSetFromMatrix(countData = cts,
    colData = coldata,
    design = ~ condition)

  featureData <- data.frame(gene=rownames(cts))
  mcols(dds) <- DataFrame(mcols(dds), featureData)
  dds <- DESeq(dds)
  res <- results(dds)

  p1 <- EnhancedVolcano(res,
    lab = rownames(res),
    x = "log2FoldChange",
    y = "pvalue",
    pCutoff = 10e-4,
    FCcutoff = 2,
    ylim = c(0, -log10(10e-12)),
    pointSize = c(ifelse(res$log2FoldChange>2, 8, 1)),
    labSize = 6.0,
    shape = c(6, 6, 19, 16),
    title = "DESeq2 results",
    subtitle = "Differential expression",
    caption = bquote(~Log[2]~ "fold change cutoff, 2; p-value cutoff, 10e-4"),
    legendPosition = "right",
    legendLabSize = 14,
    col = c("grey30", "forestgreen", "royalblue", "red2"),
    colAlpha = 0.9,
    drawConnectors = TRUE,
    hline = c(10e-8),
    widthConnectors = 0.5)

  p1


## ----ex15, fig.height = 9, fig.width = 12, fig.cap = "Highlighting key variabvles via custom point sizes."----

  p1 <- EnhancedVolcano(res,
    lab = rownames(res),
    x = "log2FoldChange",
    y = "pvalue",
    pCutoff = 10e-4,
    FCcutoff = 2,
    ylim = c(0, -log10(10e-12)),
    pointSize = c(ifelse(res$log2FoldChange>2, 8, 1)),
    labSize = 6.0,
    shape = c(6, 6, 19, 16),
    title = "DESeq2 results",
    subtitle = "Differential expression",
    caption = bquote(~Log[2]~ "fold change cutoff, 2; p-value cutoff, 10e-4"),
    legendPosition = "right",
    legendLabSize = 14,
    colAlpha = 0.9,
    colGradient = c('red3', 'royalblue'),
    drawConnectors = TRUE,
    hline = c(10e-8),
    widthConnectors = 0.5)

  p1


## ----ex16, fig.height = 9, fig.width = 12, fig.cap = "Custom axis tick marks"----

  p1 +
    ggplot2::coord_cartesian(xlim=c(-6, 6)) +
    ggplot2::scale_x_continuous(
      breaks=seq(-6,6, 1))


## -----------------------------------------------------------------------------

sessionInfo()


