Added plotly visuals + reworked log2FC calc + expanded pathway file

.Rbuildignore

@@ -15,3 +15,5 @@
 ^R/do_anova_2w\.R$
 ^R/plot_2w_anvova\.R$
 ^R/two_way_anova\.R$
+^.*\.Rproj$
+^\.Rproj\.user$

DESCRIPTION

@@ -39,15 +39,17 @@ Imports:
     ggrepel,
     utils,
     rlang,
-    methods,
     data.table,
     S4Vectors,
-    qvalue
+    viridis,
+    viridisLite,
+    plotly,
+    limma
 Description: The 'MetAlyzer' S4 object provides methods to read and reformat metabolomics data for convenient data handling, statistics and downstream analysis. The resulting format corresponds to input data of the Shiny app 'MetaboExtract' (<https://www.metaboextract.shiny.dkfz.de/MetaboExtract/>).
 License: GPL-3
 Encoding: UTF-8
 Language: en-US
-RoxygenNote: 7.2.3
+RoxygenNote: 7.3.1
 Suggests:
     rmarkdown,
     knitr

NAMESPACE

@@ -16,6 +16,9 @@ export(metalyzer_colors)
 export(pathway)
 export(plot_log2FC)
 export(plot_network)
+export(plotly_network)
+export(plotly_scatter)
+export(plotly_vulcano)
 export(polarity)
 export(renameMetaData)
 export(summarizeConcValues)
@@ -25,9 +28,17 @@ import(SummarizedExperiment)
 import(dplyr)
 import(ggplot2)
 import(ggrepel)
+import(limma)
+import(viridis)
+import(viridisLite)
 importFrom(data.table,":=")
+importFrom(plotly,add_annotations)
+importFrom(plotly,ggplotly)
+importFrom(plotly,layout)
+importFrom(plotly,plot_ly)
 importFrom(rlang,.data)
 importFrom(stats,aov)
-importFrom(stats,lm)
+importFrom(stats,model.matrix)
+importFrom(stats,na.omit)
 importFrom(tibble,deframe)
 importFrom(tibble,rownames_to_column)

R/MetAlyzer_fpaths.R

@@ -76,5 +76,5 @@ polarity <- function() {
 #' @examples
 #' fpath <- pathway()
 pathway <- function() {
-  system.file("extdata", "pathway.xlsx", package = "MetAlyzer")
+  system.file("extdata", "Pathway_130524.xlsx", package = "MetAlyzer")
 }

R/calculate_log2FC.R

@@ -1,7 +1,7 @@
 #' @title Calculate log2 fold change
 #'
-#' @description This function calculates the log2 fold change of two groups from
-#' plotting_data.
+#' @description This function calculates log2(FC), p-values, and adjusted p-values
+#' of the data using limma.
 #' @param metalyzer_se A Metalyzer object
 #' @param categorical A column specifying the two groups
 #' @param impute_perc_of_min A numeric value below 1
@@ -11,8 +11,10 @@
 #'
 #' @import dplyr
 #' @import SummarizedExperiment
+#' @import limma
 #' @importFrom rlang .data
 #' @importFrom data.table :=
+#' @importFrom stats model.matrix
 #' @export
 #'
 #' @examples
@@ -36,19 +38,6 @@ calculate_log2FC <- function(metalyzer_se,
                              categorical,
                              impute_perc_of_min = 0.2,
                              impute_NA = FALSE) {
-  ## Check for qvalue and BiocManager installation
-  # installed_packages <- utils::installed.packages()[, "Package"]
-  # if (! "qvalue" %in% installed_packages) {
-  #   if (! "BiocManager" %in% installed_packages) {
-  #     cat("Info: Installing package 'BiocManager':\n")
-  #     utils::install.packages("BiocManager")
-  #     cat("\n")
-  #   }
-  #   cat("Info: Installing package 'qvalue':\n")
-  #   BiocManager::install("qvalue", ask = FALSE, type = "binary")
-  #   cat("\n")
-  # }
-
 
   ## Create a new dataframe to calculate the log2FC
   if (!categorical %in% colnames(metalyzer_se@metadata$aggregated_data)) {
@@ -59,86 +48,50 @@ calculate_log2FC <- function(metalyzer_se,
   metalyzer_se <- data_imputation(metalyzer_se, impute_perc_of_min, impute_NA)
   metalyzer_se <- data_transformation(metalyzer_se)
 
-  df <- metalyzer_se@metadata$aggregated_data %>%
-    ungroup(all_of(categorical)) %>%
-    mutate(Value = .data$log2_Conc,
-        Group = !!sym(categorical)) %>%
-    select(.data$Metabolite,
-           .data$Class,
-           .data$Group,
-           .data$Value)
+  aggregated_data <- metalyzer_se@metadata$aggregated_data  %>%
+    dplyr::mutate(LogConcentration = log2(.data$Concentration))
+    # Prepare abundance data
+    feat_data <- dplyr::ungroup(aggregated_data) %>%
+      dplyr::select(.data$Metabolite, .data$ID, .data$LogConcentration) %>%
+      tidyr::pivot_wider(names_from = 'Metabolite', values_from = 'LogConcentration')
 
-  ## Check if already factor
-  group_vec <- df$Group
-  if (!methods::is(group_vec, "factor")) {
-    group_vec <- factor(group_vec)
-    df$Group <- group_vec
-    cat("Warning: No order was given for categorical!\n")
-  } else {
-    group_vec <- droplevels(group_vec)
-  }
-  group_levels <- levels(group_vec)
-  if (length(group_levels) > 2) {
-    cat("Warning: More than two levels were given! Dropping",
-        paste(group_levels[3:length(group_levels)], collapse = ", "), "\n")
-  }
-  cat("Info: Calculating log2 fold change from ", group_levels[1], " to ",
-      group_levels[2], " (column: ", categorical, ").\n", sep = "")
-  df <- filter(df, .data$Group %in% group_levels[1:2])
-  df$Group <- droplevels(df$Group)
+    # Prepare sample metadata of interest
+    smp_metadata <- colData(metalyzer_se) %>%
+      tibble::as_tibble(rownames = 'ID')
 
-  ## Check for further grouping
-  grouping_vars <- as.character(groups(df))
+    # Use original column names whose spaces are not replaced with '.'
+    colnames(smp_metadata) <- c('ID', colnames(colData(metalyzer_se)))
+    smp_metadata <- dplyr::select(smp_metadata, .data$ID, all_of(categorical))
 
-  if (!"Metabolite" %in% grouping_vars) {
-    grouping_vars[length(grouping_vars)+1] <- "Metabolite"
-  }
-
-  ## Calculate log2FC and p-val
-  cat("Info: Calculating log2 fold change groupwise (",
-      paste(grouping_vars, collapse = " * "),
-      ") using a linear model...  ", sep = "")
-  options(warn = -1)
-  change_df <- df %>%
-    group_modify(~ apply_linear_model(df = .x)) %>%
-    ungroup(.data$Metabolite) %>%
-    mutate(qval = qvalue::qvalue(.data$pval, pi0 = 1)$qvalues)
-  options(warn = 0)
-  cat("finished!\n")
-
-  metalyzer_se@metadata$log2FC <- change_df
-  return(metalyzer_se)
-}
+    # Combine abundance data and sample metadata to ensure matched information
+    combined_data <- dplyr::left_join(feat_data, smp_metadata, by = 'ID')
 
-#' @title Calculate log2 fold change
-#'
-#' @description This function applies a linear model to calculate the log2 fold change and
-#' its significance
-#' @param df A subset data frame
-#' @param ...
-#'
-#' @import dplyr
-#' @importFrom stats lm
-#' @importFrom rlang .data
-#'
-#' @keywords internal
-apply_linear_model <- function(df, ...) {
-  class <- df$Class[1]
-  df <- df %>%
-    filter(!is.na(.data$Value)) %>%
-    droplevels()
-  if (length(levels(df$Group)) != 2) {
-    l2fc <- NA
-    pval <- NA
-  } else {
-    fit1 <- stats::lm(Value ~ Group, data = df)
-    l2fc <- fit1$coefficients[2]
-    fit_dim <- dim(summary(fit1)$coefficients)
-    pval <- summary(fit1)$coefficients[fit_dim[1],fit_dim[2]]
-  }
-  output_df <- data.frame(Class = class,
-                          log2FC = l2fc,
-                          pval = pval,
-                          row.names = NULL)
-  return(output_df)
+    # Retrieve data matrix and sample metadata from combined data to conduct limma
+    data_mat <- combined_data[, seq_len(ncol(feat_data))] %>%
+      tibble::column_to_rownames('ID') %>%
+      t()
+    group_vec <- combined_data[, ncol(feat_data)+1, drop = T]
+
+    # Sanity check if specified categorical can split data into two groups
+    if (length(unique(group_vec)) != 2) {
+      stop("The specified categorical cannot split data into two groups.")
+    }
+
+    ## Compute log2(FC), p-values, and adjusted p-values using limma
+    design <- model.matrix(~ group_vec)
+    fit <- limma::lmFit(data_mat, design = design)
+    fit <- limma::eBayes(fit)
+    log2FCRes <- limma::topTable(fit, coef = 2, number = Inf) %>%
+      tibble::rownames_to_column('Metabolite') %>%
+      dplyr::select(.data$Metabolite, .data$logFC, .data$P.Value, .data$adj.P.Val) %>%
+      dplyr::rename(log2FC = .data$logFC, pval = .data$P.Value, qval = .data$adj.P.Val)
+    # Combined all information into a table
+    group_info <- combined_data[, c(1, ncol(feat_data)+1)]
+    log2FCTab <- dplyr::left_join(aggregated_data, group_info, by = 'ID') %>%
+      dplyr::left_join(log2FCRes, by = 'Metabolite') %>%
+      dplyr::select(.data$Metabolite, .data$Class, .data$log2FC, .data$pval, .data$qval) %>%
+      dplyr::distinct(.data$Metabolite, .keep_all = TRUE)
+    metalyzer_se@metadata$log2FC <- log2FCTab
+
+    return(metalyzer_se)
 }