Author: Frederik Ziebell

# Creating The Example Data Set

#### Frederik Ziebell

Here we quickly show how the data objects of the example data set can be reproduced.

# Load Packages

    library("recount3")
    library("DESeq2")
    library("dplyr")
    library("stringr")
    library("SummarizedExperiment")
    library("tidyr")
    library("tibble")
    library("magrittr")

# Download GSE89888 via recount3 and format data

    # download data recount3
    proj_info <- available_projects() %>%
      filter(project == "SRP093386")
    se <- create_rse(proj_info)

    count_mat <- compute_read_counts(se)
    rownames(count_mat) <- str_replace(rownames(count_mat), "\\.[:number:]+$", "")

    meta <- colData(se) %>%
      as_tibble() %>%
      separate(sra.sample_title, into = c("cell_line", "treatment", "mutation", "replicate"), sep = "-") %>%
      select(cell_line, treatment, mutation, replicate)

    colnames(count_mat) <- paste0(meta$treatment, "_", meta$mutation, "_", meta$replicate)

    # subset to cell line T47D
    count_mat <- count_mat[, meta$cell_line == "T47D"]
    meta <- meta[meta$cell_line == "T47D", c("treatment", "mutation", "replicate")]

    T47D <- make_dds(count_mat, meta, ah_record = "AH89426")
    T47D <- T47D[rowSums(assay(T47D))>0,]

    # round some numeric data to reduce the size of the data object
    rowData(T47D)$gc_content <- round(rowData(T47D)$gc_content,1)

# Differential testing

    dds <- T47D
    dds <- filter_genes(dds, min_count = 5, min_rep = 4)
    dds$mutation <- as.factor(dds$mutation)
    dds$treatment <- as.factor(dds$treatment)
    design(dds) <- ~ mutation + treatment

    # to not run DESeq2 in the main vignette,
    # wo pre-compute the dispersion plot and diff testing results
    dds <- DESeq(dds, parallel=T)

    png(filename="disp_ests.png", width=7, height=5, units="in", res=200)
    plotDispEsts(dds)
    dev.off()

    T47D_diff_testing <- lfcShrink(dds, coef = "mutation_WT_vs_D538G", lfcThreshold = log2(1.5), type = "normal", parallel = TRUE)
    T47D_diff_testing$stat <- NULL
    T47D_diff_testing$lfcSE <- NULL
    T47D_diff_testing$pvalue <- NULL