Acute Tunicamycin mouse model
Christian H. Holland
2020-12-19
Last updated: 2020-12-20
Checks: 7 0
Knit directory: meta-liver/
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Introduction
Here we analysis a mouse model of Tunicamycin induced acute liver damage.
Libraries and sources
These libraries and sources are used for this analysis.
library(mouse4302.db)
library(tidyverse)
library(tidylog)
library(here)
library(oligo)
library(annotate)
library(limma)
library(biobroom)
library(janitor)
library(AachenColorPalette)
library(cowplot)
library(lemon)
library(patchwork)
options("tidylog.display" = list(print))
source(here("code/utils-microarray.R"))
source(here("code/utils-utils.R"))
source(here("code/utils-plots.R"))Definition of global variables that are used throughout this analysis.
# i/o
data_path <- "data/mouse-acute-tunicamycin"
output_path <- "output/mouse-acute-tunicamycin"
# graphical parameters
# fontsize
fz <- 9Data processing
Load .CEL files and quality control
The array quality is controlled based on the relative log expression values (RLE) and the normalized unscaled standard errors (NUSE).
# load cel files and check quality
platforms <- readRDS(here("data/annotation/platforms.rds"))
raw_eset <- list.celfiles(here(data_path), listGzipped = T, full.names = T) %>%
read.celfiles() %>%
ma_qc()
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-tunicamycin/GSM740952.CEL.gz
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-tunicamycin/GSM740953.CEL.gz
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-tunicamycin/GSM740954.CEL.gz
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-tunicamycin/GSM740955.CEL.gz
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-tunicamycin/GSM740956.CEL.gz
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-tunicamycin/GSM740957.CEL.gz
#> Reading in : /Users/cholland/Projects/meta-liver/data/mouse-acute-tunicamycin/GSM740958.CEL.gzNormalization and probe annotation
Probe intensities are normalized with the rma() function. Probes are annotated with MGI symbols.
eset <- rma(raw_eset)
#> Background correcting
#> Normalizing
#> Calculating Expression
# annotate microarray probes with mgi symbols
expr <- ma_annotate(eset, platforms)
colnames(expr) = str_remove(colnames(expr), ".CEL.gz")
# gene "BC001981" has a constant value across all samples and must be thus removed
constant_genes = which(apply(expr, 1, var) == 0)
expr = expr[-constant_genes,]
# save normalized expression
saveRDS(expr, here(output_path, "normalized_expression.rds"))Build meta data
Meta information are parsed from the sample names.
# build meta data
meta = colnames(expr) %>%
enframe(name = NULL, value = "sample") %>%
mutate(group = c(rep("treated", 4), rep("control", 3))) %>%
mutate(group = factor(group, levels = c("control", "treated")))
#> mutate: new variable 'group' (character) with 2 unique values and 0% NA
#> mutate: converted 'group' from character to factor (0 new NA)
# save meta data
saveRDS(meta, here(output_path, "meta_data.rds"))Exploratory analysis
PCA of normalized data
PCA plot of normalized expression data contextualized based on the treatment. Only the top 1000 most variable genes are used as features.
expr <- readRDS(here(output_path, "normalized_expression.rds"))
meta <- readRDS(here(output_path, "meta_data.rds"))
pca_result <- do_pca(expr, meta, top_n_var_genes = 1000)
#> left_join: added one column (group)
#> > rows only in x 0
#> > rows only in y (0)
#> > matched rows 7
#> > ===
#> > rows total 7
saveRDS(pca_result, here(output_path, "pca_result.rds"))
plot_pca(pca_result, feature = "group") +
my_theme()
Differential gene expression analysis
Running limma
Differential gene expression analysis via limma with the aim to identify the effect of Tunicamycin intoxication.
# load expression and meta data
expr <- readRDS(here(output_path, "normalized_expression.rds"))
meta <- readRDS(here(output_path, "meta_data.rds"))
stopifnot(colnames(expr) == meta$sample)
# build design matrix
design = model.matrix(~0+group, data=meta)
rownames(design) = meta$sample
colnames(design) = levels(meta$group)
# define contrasts
contrasts = makeContrasts(
treat_vs_ctrl = treated - control,
levels = design
)
limma_result = run_limma(expr, design, contrasts) %>%
assign_deg()
#> select: renamed 3 variables (contrast, logFC, pval) and dropped one variable
#> group_by: one grouping variable (contrast)
#> mutate (grouped): new variable 'fdr' (double) with 13,527 unique values and 0% NA
#> ungroup: no grouping variables
#> mutate: new variable 'regulation' (character) with 3 unique values and 0% NA
#> mutate: converted 'regulation' from character to factor (0 new NA)
saveRDS(limma_result, here(output_path, "limma_result.rds"))Volcano plots
Volcano plots visualizing the effect of Tunicamycin on gene expression.
df <- readRDS(here(output_path, "limma_result.rds"))
df %>%
plot_volcano() +
my_theme(grid = "y", fsize = fz)
#> rename: renamed one variable (p)
sessionInfo()
#> R version 4.0.2 (2020-06-22)
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#> Running under: macOS Mojave 10.14.5
#>
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#> LAPACK: /Library/Frameworks/R.framework/Versions/4.0/Resources/lib/libRlapack.dylib
#>
#> locale:
#> [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
#>
#> attached base packages:
#> [1] parallel stats4 stats graphics grDevices datasets utils
#> [8] methods base
#>
#> other attached packages:
#> [1] pd.mouse430.2_3.12.0 DBI_1.1.0 RSQLite_2.2.1
#> [4] patchwork_1.1.1 lemon_0.4.5 cowplot_1.1.0
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#> [10] broom_0.7.3 limma_3.44.3 annotate_1.66.0
#> [13] XML_3.99-0.5 oligo_1.52.1 Biostrings_2.56.0
#> [16] XVector_0.28.0 oligoClasses_1.50.4 here_1.0.1
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#> [25] tidyr_1.1.2 tibble_3.0.4 ggplot2_3.3.2
#> [28] tidyverse_1.3.0 mouse4302.db_3.2.3 org.Mm.eg.db_3.11.4
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