---

name: global-methylation-profile description: This skill performs genome-wide DNA methylation profiling. It supports single-sample and multi-sample workflows to compute methylation density distributions, genomic feature distribution of the methylation profile, and sample-level clustering/PCA. Use it when you want to systematically characterize global methylation patterns from WGBS or similar per-CpG methylation call files.

Global DNA Methylation Profiling

Overview

Main steps include:

• Refer to the Inputs & Outputs section to check available inputs and design the output structure.
• Always prompt user for genome assembly used.
• Always prompt user for which columns are methylation fraction/percent and coverage and strand.
• Analyze the genomic feature distribution of methylations for each sample.
• Compute and visualize genome-wide methylation density distributions.
• For multi-sample datasets, prepare the matrix of methylation data.
• Perform PCA and hierarchical clustering to assess sample similarity based on global methylation.
• Never use MCP tools in this skill, use R scripts instead.

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When to use this skill

Use the global-methylation-profiling skill when you want to:

• Characterize global DNA methylation status of one or multiple samples (e.g. normal vs tumor, different cell types).
• Compare broad methylation patterns across samples:
  • Are some samples globally hypo-/hyper-methylated?
  • Are certain chromosomes or genomic regions more strongly affected?
• Explore genomic feature of your methylation dataset (e.g. promoter hypomethylation, gene body hypermethylation).
• Perform unsupervised clustering/PCA to see if samples separate by condition based on genome-wide methylation patterns.

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Inputs & Outputs

Inputs

<sample.bed

Outputs

global_methylation_profile/
  stats/
    summary_statistics.tsv
    ...
  plots/
    sample1_genomic_feature_pie.pdf
    sample2_genomic_feature_pie.pdf
    ... # Other samples
    allSamples_methylation_density_overlay.pdf
    PCA_scatterplot.pdf
    sample_correlation_heatmap.pdf
    ...
  logs/
  temp/ # all the temp files

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Decision Tree

Step 1: Prepare the sample meta data

library(methylKit)
# Example input: Bismark coverage files (chr, start, end, numCs, numTs, strand)
file.list <- list(
  "sample1.cov",
  "sample2.cov",
  "sample3.cov"
)

sample.id <- list("S1", "S2", "S3")
treatment <- c(0, 1, 1)  # e.g. 0 = control, 1 = treated

# Read methylation data
myobj <- methRead(
  location = file.list,
  sample.id = sample.id,
  assembly  = "hg38", # provided by user
  treatment = treatment,
  context   = "CpG",
  pipeline = list(
    fraction = FALSE,  # percMeth is 0–100, fraction is 0-1, depend on inputs
    chr.col = 1,
    start.col = 2,
    end.col = 3,
    strand.col = 6, # provided by user
    coverage.col = 10, # provided by user
    freqC.col = 11 # provided by user
  )
)

# Optional filtering: remove low / extremely high coverage CpGs
filtered.myobj <- filterByCoverage(
  myobj,
  lo.count = 10, lo.perc = NULL,
  hi.count = 99.9, hi.perc = TRUE
)

# Unite CpGs across samples (common CpG sites)
meth <- unite(filtered.myobj, destrand = TRUE)

Step 2: Analyze Genomic Feature Distribution of CpGs

Annotate CpGs with genomic features (promoter, exon, intron, intergenic, etc.) with genomation and summarize where CpGs (or methylated CpGs) are located for each sample

library(genomation)
library(TxDb.Hsapiens.UCSC.hg38.knownGene) # depend on user inputs
txdb <- TxDb.Hsapiens.UCSC.hg38.knownGene
# exons
exons <- unlist(exonsBy(txdb))
names(exons) <- NULL
mcols(exons)$type <- "exon"
# introns
introns <- unlist(intronsByTranscript(txdb))
names(introns) <- NULL
mcols(introns)$type <- "intron"
# promoters
promoters.gr <- promoters(txdb, upstream = 2000, downstream = 200)
names(promoters.gr) <- NULL
mcols(promoters.gr)$type <- "promoter"
# TSS（1bp）
TSSes <- promoters(txdb, upstream = 1, downstream = 1)
names(TSSes) <- NULL
mcols(TSSes)$type <- "TSS"
# 3'UTR
utr3 <- unlist(threeUTRsByTranscript(txdb))
names(utr3) <- NULL
mcols(utr3)$type <- "UTR3"
# 5'UTR
utr5 <- unlist(fiveUTRsByTranscript(txdb))
names(utr5) <- NULL
mcols(utr5)$type <- "UTR5"

gene.obj <- GRangesList(
  promoters = promoters.gr,
  exons     = exons,
  introns   = introns,
  TSSes     = TSSes
  UTR5      = utr5,
  UTR3      = utr3,
  ... # other features
)

for (i in seq_along(filtered.myobj)) {
  sample_id <- filtered.myobj[[i]]@sample.id
  cpg.gr <- as(filtered.myobj[[i]], "GRanges")
  ann.gene <- annotateWithGeneParts(cpg.gr, gene.obj)
  feature.summary <- getTargetAnnotationStats(ann.gene, percentage = TRUE)
  out_tab <- as.data.frame(feature.summary)
  write.table(
    out_tab,
    file      = file.path(plot_dir, paste0(sample_id, "_feature_annotation_stats.tsv")),
    sep       = "\t", quote = FALSE, row.names = FALSE
  )
  pdf(file.path(plot_dir, paste0(sample_id, "_genomic_feature_distribution.pdf")))
  plotTargetAnnotation(
    ann.gene,
    main = paste("Genomic feature distribution of CpGs -", sample_id)
  )
  dev.off()
}

Step 3: Compute & visualize genome-wide methylation density distributions

# Convert to percent methylation matrix: rows = CpGs, cols = samples
meth.mat <- percMethylation(meth)  # values 0–100

df.long <- reshape2::melt(
  as.data.frame(meth.mat),
  variable.name = "Sample",
  value.name   = "Methylation"
)

ggplot(df.long, aes(x = Methylation, color = Sample)) +
  geom_density() +
  theme_bw() +
  xlab("Percent methylation") +
  ggtitle("Genome-wide methylation density across samples")

Step 4: PCA & Hierarchical Clustering of Multi-sample Methylation

• Use CpG methylation profiles across samples to assess sample similarity and batch effects.

# Meth matrix: rows = CpGs, cols = samples (0–100)
meth.mat <- percMethylation(meth)

# (Optional) Filter CpGs by variability
cpg.sd <- apply(meth.mat, 1, sd, na.rm = TRUE)
keep.var <- cpg.sd > 0
meth.var <- meth.mat[keep.var, ]
if (sum(keep.var) > 10000) {
  keep.idx <- order(cpg.sd[keep.var], decreasing = TRUE)[1:10000]
  meth.var <- meth.var[keep.idx, ]
}

# Z-score transformation (per CpG) – helps clustering
meth.scaled <- t(scale(t(meth.var)))  # rows scaled

pca <- prcomp(t(meth.scaled), center = FALSE, scale. = FALSE)
pca.df <- data.frame(
  Sample = colnames(meth.scaled),
  PC1 = pca$x[, 1],
  PC2 = pca$x[, 2],
  Treatment = factor(treatment, labels = c("Control", "Treatment"))
)

ggplot(pca.df, aes(x = PC1, y = PC2, color = Treatment, label = Sample)) +
  geom_point(size = 3) +
  geom_text(vjust = -1) +
  theme_bw() +
  ggtitle("PCA of global CpG methylation") +
  xlab(paste0("PC1 (", round(summary(pca)$importance[2, 1] * 100, 1), "%)")) +
  ylab(paste0("PC2 (", round(summary(pca)$importance[2, 2] * 100, 1), "%)"))

dist.samples <- dist(t(meth.scaled), method = "euclidean")
hc <- hclust(dist.samples, method = "complete")

plot(hc, main = "Hierarchical clustering of samples (methylation)",
     xlab = "", sub = "")

cor.samples <- cor(meth.var, use = "pairwise.complete.obs")

pheatmap(cor.samples,
         clustering_method = "complete",
         main = "Sample correlation based on CpG methylation")