sample_check.R

#### This script is for analysis in R AFTER COUNT GENERATION AND CREATING A METADATA FILE

#Sample quality check

# load required raw count data and metadata                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   metadata <- readRDS(here::here("samples.metadata.RDS"))
reads_count <- readRDS(file = here::here("read.counts.RDS"))
samples.metadata.clean <- readRDS(here::here("samples.metadata.RDS"))

# Create a DGEList object
counts <- DGEList(reads_count, group = samples.metadata$Status)

# check the number of genes with no expression in all samples
table(rowSums(counts$counts==0)==45)

# Filtering non-expressed and lowly-expressed genes
keep.exprs <- filterByExpr(counts, group=samples.metadata$Status)
filtered.counts <- counts[keep.exprs,, keep.lib.sizes=FALSE]

# obtain logCPM unnormalized for plotting purposes
# Here, the norm.factors value is 1 for all samples
logcpm.unnorm.counts <- cpm(filtered.counts, log = TRUE, prior.count = 2, normalized.lib.sizes = TRUE)

# Normalize for composition bias using TMM
filtered.counts <- calcNormFactors(filtered.counts, method = 'TMM')

# Convert counts per million per gene to log counts per million for further downstream analysis.
logcpm.norm.counts <- cpm(filtered.counts, log = TRUE, prior.count = 2, normalized.lib.sizes = TRUE)

# save outputs for later analysis
saveRDS(filtered.counts, here::here("filtered.counts.RDS"))
saveRDS(logcpm.unnorm.counts, here::here("logcpm.unnorm.counts.RDS"))
saveRDS(logcpm.norm.counts, here::here("logcpm.norm.counts.RDS"))