@@ -23,6 +23,7 @@ Imports:

     AnnotationDbi,

     bs4Dash,

     colorspace,

+	colourpicker,

     ComplexHeatmap,

     dendextend,

     DESeq2,

@@ -18,6 +18,7 @@ export(geneinfo_2_html)

 export(get_aggrscores)

 export(get_expression_values)

 export(ggs_graph)

+export(ggs_volcano)

 export(go_2_html)

 export(gs_alluvial)

 export(gs_dendro)

@@ -92,6 +93,7 @@ importFrom(bs4Dash,bs4ValueBoxOutput)

 importFrom(bs4Dash,renderbs4InfoBox)

 importFrom(bs4Dash,renderbs4ValueBox)

 importFrom(colorspace,rainbow_hcl)

+importFrom(colourpicker,colourInput)

 importFrom(dendextend,branches_attr_by_clusters)

 importFrom(dendextend,set)

 importFrom(dplyr,"%>%")

@@ -13,6 +13,7 @@

 #' bs4ValueBoxOutput renderbs4InfoBox renderbs4ValueBox

 #' bs4TabPanel bs4TabCard

 #' @importFrom colorspace rainbow_hcl

+#' @importFrom colourpicker colourInput

 #' @importFrom ComplexHeatmap Heatmap HeatmapAnnotation draw

 #' @importFrom dendextend branches_attr_by_clusters set

 #' @importFrom DESeq2 vst counts estimateSizeFactors normalizationFactors sizeFactors

@@ -17,7 +17,7 @@

 #' @param FDR Numeric value, specifying the significance level for thresholding

 #' adjusted p-values. Defaults to 0.05.

 #' @param color Character string to specify color of filtered points in the plot.

-#'  Default color is red.

+#'  Default color is #0092AC.

 #' @param volcano_labels Integer, maximum number of labels for the gene sets to be

 #' plotted as labels on the volcano scatter plot.

 #' @param plot_title Character string, to specify the title of the plot,

@@ -39,7 +39,6 @@

 #' rownames(dds_macrophage) <- substr(rownames(dds_macrophage), 1, 15)

 #' dds_macrophage <- estimateSizeFactors(dds_macrophage)

 #'

-#' vst_macrophage <- vst(dds_macrophage)

 #'

 #' # annotation object

 #' anno_df <- data.frame(

@@ -61,8 +60,7 @@

 #' res_enrich <- shake_topGOtableResult(topgoDE_macrophage_IFNg_vs_naive)

 #' res_enrich <- get_aggrscores(res_enrich, res_de, anno_df)

 #'

-#' ggs_volcano(vst_macrophage,

-#'            res_de,

+#' ggs_volcano(res_de,

 #'            res_enrich,

 #'            anno_df,

 #'            geneset_id = res_enrich$gs_id[1]

@@ -76,7 +74,7 @@ ggs_volcano <- function(res_de,

                        genelist = NULL,

                        FDR = 0.05,

                        color = "red",

-                       volcano_labels = 10,

+                       volcano_labels = 25,

                        plot_title = NULL

                        ) {

@@ -96,27 +94,48 @@ ggs_volcano <- function(res_de,

       thisset_members_ids <- annotation_obj$gene_id[match(thisset_members, annotation_obj$gene_name)]

     }

   }else {

-    # overridable via a list

-    if (!all(genelist %in% rownames(se))) {

-      not_there <- genelist[!(genelist %in% rownames(se))]

+    # overwritable via a list

+    if (!all(genelist %in% res_de@rownames)) {

+      not_there <- genelist[!(genelist %in% res_de@rownames)]

       warning("Some of the provided gene ids were not found in the SummarizedExperiment",

               "\nNot found: ", not_there)

     }

-    thisset_members_ids <- intersect(rownames(se), genelist)

+    thisset_members_ids <- intersect(res_de@rownames, genelist)

     thisset_name <- "Custom list"

   }

   thisset_members_ids

   thisset_members

+  complete_genes_ids <- res_de@rownames

+  complete_genes <- annotation_obj$gene_name[match(complete_genes_ids, annotation_obj$gene_id)]

+  padj_complete <- res_de[complete_genes_ids, "padj"]

+  filter_info_complete <- sapply(padj_complete, function(x) x <= FDR)

+  padj_complete <- sapply(padj_complete, function(x) -log10(x))

+  log2FoldChange_complete <- res_de[complete_genes_ids, "log2FoldChange"]

+  gene_set_belong <- complete_genes_ids %in% thisset_members_ids

+  

+  filter_info_complete <- filter_info_complete & gene_set_belong

+

+  

+  thisset_complete_data <- data.frame(complete_genes_ids, padj_complete, log2FoldChange_complete, filter_info_complete, gene_set_belong)

+  colnames(thisset_complete_data) <- c("genes", "logTransformedpvalue", "log2FoldChange", "significant", "belonging")

+  

+  

+  # Prepare plotting

+  volcano_df_complete <- thisset_complete_data

+  volcano_df_complete$genes_name <- complete_genes

+  max_x <- max(abs(range(thisset_complete_data["log2FoldChange"])))

+  limit_x <- max_x * c(-1, 1)

+  

   # retrieve adjusted pvalues and log2Fold Change for genes

-  padj <- res_de[thisset_members_ids, "padj"]

-  padjlog <- sapply(padj, function(x) -log10(x))

-  log2FoldChange <- res_de[thisset_members_ids, "log2FoldChange"]

-  filter_info <- sapply(padj, function(x) x <= FDR)

+  #padj <- res_de[thisset_members_ids, "padj"]

+  #padjlog <- sapply(padj, function(x) -log10(x))

+  #log2FoldChange <- res_de[thisset_members_ids, "log2FoldChange"]

+  #filter_info <- sapply(padj, function(x) x <= FDR)

   # Prepare data to plot

-  thisset_data <- data.frame(thisset_members_ids, padj, log2FoldChange, padjlog, filter_info)

+  #thisset_data <- data.frame(thisset_members_ids, padj, log2FoldChange, padjlog, filter_info)

   if (is.null(plot_title)) {

@@ -125,30 +144,55 @@ ggs_volcano <- function(res_de,

     title <- plot_title

   }

-  colnames(thisset_data) <- c("genes", "pvalue", "log2FoldChange", "logTransformedpvalue", "significant")

+ # colnames(thisset_data) <- c("genes", "pvalue", "log2FoldChange", "logTransformedpvalue", "significant")

   # Prepare plotting

-  volcano_df <- thisset_data

-  volcano_df$genes_name <- thisset_members

-  max_x <- max(abs(range(thisset_data["log2FoldChange"])))

-  limit_x <- max_x * c(-1, 1)

+ # volcano_df <- thisset_data

+  #volcano_df$genes_name <- thisset_members

+  #max_x <- max(abs(range(thisset_data["log2FoldChange"])))

+  #limit_x <- max_x * c(-1, 1)

   # Plot data

-   p <- ggplot(volcano_df, aes(x = log2FoldChange, y = logTransformedpvalue, label = genes_name)) +

-    geom_point(aes(color = significant)) +

+  #p <- ggplot(volcano_df, aes(x = log2FoldChange, y = logTransformedpvalue, label = genes_name)) +

+  #geom_point(aes(color = significant)) +

+  #  labs(x = "log2Fold Change",

+  #       y = "-log10 p-value", color = "p-value <= FDR") + 

+  #  scale_x_continuous(limits = limit_x) +

+  #  scale_color_manual(labels = c("significant", "not significant"), 

+  #                     breaks = c("TRUE", "FALSE"), values = c(color, "grey25")) +

+  #  theme(legend.title = element_text(size = 11, face = "bold"), legend.text = element_text(size = 10)) +

+  #  geom_text_repel()

+  

+  p <- ggplot(volcano_df_complete, aes_string(x = "log2FoldChange", y = "logTransformedpvalue")) +

+    geom_point(aes_string(color = "significant", alpha = "belonging")) +

     labs(x = "log2Fold Change",

-         y = "-log10 p-value", color = "p-value <= FDR") + 

+         y = "-log10 p-value",

+         color = "p-value <= FDR") + 

     scale_x_continuous(limits = limit_x) +

     scale_color_manual(labels = c("significant", "not significant"), 

-                       breaks = c("TRUE", "FALSE"), values = c(color, "grey25")) +

-    theme(legend.title = element_text(size = 11, face = "bold"), legend.text = element_text(size = 10)) +

-    geom_text_repel()

+                       breaks = c("TRUE", "FALSE"),

+                       values = c(color, "grey25")) +

+    scale_alpha_manual(breaks = c("TRUE", "FALSE"),

+                       values = c(1, 1/10)) +

+    theme_bw() +

+    theme(legend.title = element_text(size = 11, face = "bold"),

+          legend.text = element_text(size = 10))  

+    

+  # adding labels to the significant points of the geneset

+  p <- p + geom_text_repel(

+    data = subset(volcano_df_complete, filter_info_complete),

+    aes_string(label = "genes_name"),

+    size = 4,

+    max.overlaps = volcano_labels)

+

+

   # handling the title

   p <- p + ggtitle(title)

+  p <- p + guides(alpha = FALSE)

   return(p)

 }

new file mode 100644

@@ -0,0 +1,97 @@

+% Generated by roxygen2: do not edit by hand

+% Please edit documentation in R/ggs_volcano.R

+\name{ggs_volcano}

+\alias{ggs_volcano}

+\title{Plot a volcano plot of the gene signature on the data}

+\usage{

+ggs_volcano(

+  res_de,

+  res_enrich,

+  annotation_obj = NULL,

+  gtl = NULL,

+  geneset_id = NULL,

+  genelist = NULL,

+  FDR = 0.05,

+  color = "red",

+  volcano_labels = 25,

+  plot_title = NULL

+)

+}

+\arguments{

+\item{res_de}{A \code{DESeqResults} object.}

+

+\item{res_enrich}{A \code{data.frame} object, storing the result of the functional

+enrichment analysis. See more in the main function, \code{\link[=GeneTonic]{GeneTonic()}}, to check the

+formatting requirements (a minimal set of columns should be present).}

+

+\item{annotation_obj}{A \code{data.frame} object with the feature annotation

+information, with at least two columns, \code{gene_id} and \code{gene_name}.}

+

+\item{gtl}{A \code{GeneTonic}-list object, containing in its slots the arguments

+specified above: \code{dds}, \code{res_de}, \code{res_enrich}, and \code{annotation_obj} - the names

+of the list \emph{must} be specified following the content they are expecting}

+

+\item{geneset_id}{Character specifying the gene set identifier to be plotted}

+

+\item{genelist}{A vector of character strings, specifying the identifiers

+contained in the row names of the \code{se} input object.}

+

+\item{FDR}{Numeric value, specifying the significance level for thresholding

+adjusted p-values. Defaults to 0.05.}

+

+\item{color}{Character string to specify color of filtered points in the plot.

+Default color is #0092AC.}

+

+\item{volcano_labels}{Integer, maximum number of labels for the gene sets to be

+plotted as labels on the volcano scatter plot.}

+

+\item{plot_title}{Character string, to specify the title of the plot,

+displayed over the heatmap. If left to \code{NULL} as by default, it tries to use

+the information on the geneset identifier provided}

+}

+\value{

+A plot returned by the \code{\link[=ggplot]{ggplot()}} function

+}

+\description{

+Plot a volcano plot for the selected gene signature on the provided data, with the possibility to compactly display also DE only genes

+}

+\examples{

+library("macrophage")

+library("DESeq2")

+library("org.Hs.eg.db")

+library("AnnotationDbi")

+

+# dds object

+data("gse", package = "macrophage")

+dds_macrophage <- DESeqDataSet(gse, design = ~line + condition)

+rownames(dds_macrophage) <- substr(rownames(dds_macrophage), 1, 15)

+dds_macrophage <- estimateSizeFactors(dds_macrophage)

+

+

+# annotation object

+anno_df <- data.frame(

+  gene_id = rownames(dds_macrophage),

+  gene_name = mapIds(org.Hs.eg.db,

+                     keys = rownames(dds_macrophage),

+                     column = "SYMBOL",

+                     keytype = "ENSEMBL"),

+  stringsAsFactors = FALSE,

+  row.names = rownames(dds_macrophage)

+)

+

+# res object

+data(res_de_macrophage, package = "GeneTonic")

+res_de <- res_macrophage_IFNg_vs_naive

+

+# res_enrich object

+data(res_enrich_macrophage, package = "GeneTonic")

+res_enrich <- shake_topGOtableResult(topgoDE_macrophage_IFNg_vs_naive)

+res_enrich <- get_aggrscores(res_enrich, res_de, anno_df)

+

+ggs_volcano(res_de,

+           res_enrich,

+           anno_df,

+           geneset_id = res_enrich$gs_id[1]

+           )

+

+}