Chapter 3 Visualizing distributions

Before choosing a normalization method, inspect data distributions.

3.1 load Some data

library(GEOquery)

## Loading required package: Biobase

## Loading required package: BiocGenerics

## Loading required package: generics

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##     order, paste, pmax, pmax.int, pmin, pmin.int, Position, rank,
##     rbind, Reduce, rownames, sapply, saveRDS, table, tapply, unique,
##     unsplit, which.max, which.min

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## Setting options('download.file.method.GEOquery'='auto')

## Setting options('GEOquery.inmemory.gpl'=FALSE)

library(knitr)
library(dplyr)

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library(tibble)
if(!require(tidyverse)){
  install.packages("tidyvers",dependencies = FALSE)
}

## Loading required package: tidyverse

## Error: package or namespace load failed for 'tidyverse':
##  .onAttach failed in attachNamespace() for 'tidyverse', details:
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##   error: there is no package called 'lubridate'

## Installing package into '/usr/local/lib/R/site-library'
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library(tidyverse)

gse <- "GSE119732"

source("./supp_functions.R")
fetch_geo_supp(gse = gse)

## Using locally cached version of supplementary file(s) GSE119732 found here:
## data/GSE119732/GSE119732_count_table_RNA_seq.txt.gz

path <- file.path("data", gse)
files <- list.files(path, pattern = "\\.txt.gz$|\\.tsv.gz$|\\.csv.gz$", 
                    full.names = TRUE, recursive = TRUE)

Raw table preview

library(readr)

safe_read <- function(file) {
  # First attempt: read as TSV
  df <- tryCatch(
    readr::read_tsv(file, show_col_types = FALSE),
    error = function(e) NULL   # catch fatal errors
  )
  
  # If read_tsv failed entirely:
  if (is.null(df)) {
    message("TSV read failed — reading as space-delimited file instead.")
    return(readr::read_table(file, show_col_types = FALSE))
  }
  
  # If read_tsv returned but with parsing issues:
  probs <- problems(df)
  if (nrow(probs) > 0) {
    message("Parsing issues detected in TSV — reading as space-delimited file instead.")
    return(readr::read_table(file, show_col_types = FALSE))
  }
  
  # If everything was fine:
  return(df)
}

x <- safe_read(files[1])


kable_head(x[, 1:min(6, ncol(x))], 5, paste(gse,": raw table preview"))

(#tab:preview_raw)GSE119732 : raw table preview

gene_id	A1	A2	A3	A4	B1
ENSG00000223972.5	0	0	0	0	0
ENSG00000227232.5	79	119	84	50	80
ENSG00000278267.1	17	10	22	19	19
ENSG00000243485.4	0	0	0	0	0
ENSG00000237613.2	0	0	0	0	0

3.2 Boxplots

library(tidyverse)

# suppose 'mat' is a gene x sample matrix (numeric)
plot_box <- function(mat, main = "", ylab = "log2(counts+1)") {
  df <- as.data.frame(mat)
  df_long <- df |>
    mutate(gene = rownames(df)) |>
    pivot_longer(-gene, names_to = "sample", values_to = "value") |>
    mutate(value = log2(value + 1))

  ggplot(df_long, aes(x = sample, y = value)) +
    geom_boxplot(outlier.size = 0.2) +
    theme_bw() +
    theme(axis.text.x = element_text(angle = 90, vjust = 0.5, hjust = 1)) +
    labs(title = main, x = "Sample", y = ylab)
}

plot_box(x[,2:ncol(x)])

3.3 Density plots

plot_density <- function(mat, main = "") {
  df <- as.data.frame(mat)
  df_long <- df |>
    mutate(gene = rownames(df)) |>
    pivot_longer(-gene, names_to = "sample", values_to = "value") |>
    mutate(value = log2(value + 1))

  ggplot(df_long, aes(x = value, colour = sample)) +
    geom_density() +
    theme_bw() +
    labs(title = main, x = "log2(counts+1)", y = "Density") +
    guides(colour = "none")
}

plot_density(x[,2:ncol(x)])