M6a differential

Version Compatibility

Reference examples tested with: ggplot2 3.5+

Before using code patterns, verify installed versions match. If versions differ:

• R: packageVersion('<pkg>') then ?function_name to verify parameters

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Differential m6A Analysis

"Find differential m6A sites between my conditions" → Identify RNA methylation changes between experimental groups by comparing MeRIP-seq IP/input ratios across conditions with statistical testing.

• R: exomePeak2::exomePeak2() with contrast design for differential peaks

exomePeak2 Differential Analysis

Goal: Identify m6A sites that differ in methylation level between experimental conditions from MeRIP-seq data.

Approach: Run exomePeak2 with a contrast design matrix comparing IP/input ratios across conditions, which accounts for GC bias and biological replicates.

library(exomePeak2)

# Define sample design
# condition: factor for comparison
design <- data.frame(
    condition = factor(c('ctrl', 'ctrl', 'treat', 'treat'))
)

# Differential peak calling
result <- exomePeak2(
    bam_ip = c('ctrl_IP1.bam', 'ctrl_IP2.bam', 'treat_IP1.bam', 'treat_IP2.bam'),
    bam_input = c('ctrl_Input1.bam', 'ctrl_Input2.bam', 'treat_Input1.bam', 'treat_Input2.bam'),
    gff = 'genes.gtf',
    genome = 'hg38',
    experiment_design = design
)

# Get differential sites
diff_sites <- results(result, contrast = c('condition', 'treat', 'ctrl'))

QNB for Differential Methylation

library(QNB)

# Requires count matrices from peak regions
# IP and input counts per sample
qnb_result <- qnbtest(
    IP_count_matrix,
    Input_count_matrix,
    group = c(1, 1, 2, 2)  # 1=ctrl, 2=treat
)

# Filter significant
# padj < 0.05, |log2FC| > 1
sig <- qnb_result[qnb_result$padj < 0.05 & abs(qnb_result$log2FC) > 1, ]

Visualization

library(ggplot2)

# Volcano plot
ggplot(diff_sites, aes(x = log2FoldChange, y = -log10(padj))) +
    geom_point(aes(color = padj < 0.05 & abs(log2FoldChange) > 1)) +
    geom_hline(yintercept = -log10(0.05), linetype = 'dashed') +
    geom_vline(xintercept = c(-1, 1), linetype = 'dashed')

Related Skills

• m6a-peak-calling - Identify peaks first
• differential-expression/de-results - Similar statistical concepts
• modification-visualization - Plot differential sites