Version Compatibility

Reference examples tested with: DESeq2 1.42+, edgeR 4.0+, ggplot2 3.5+, scanpy 1.10+

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 miRNA Expression

"Find differentially expressed miRNAs between my conditions" → Perform statistical testing on miRNA count matrices to identify miRNAs with significant expression changes, accounting for small RNA-specific normalization considerations.

• R:

  DESeq2::DESeq()

  or

  edgeR::glmQLFTest()

  on miRNA count data

Load miRNA Count Data

library(DESeq2)

# Load miRge3 or miRDeep2 counts
counts <- read.csv('miR.Counts.csv', row.names = 1)

# Create sample metadata
coldata <- data.frame(
    sample = colnames(counts),
    condition = factor(c('control', 'control', 'treated', 'treated')),
    row.names = colnames(counts)
)

DESeq2 Analysis

Goal: Identify miRNAs with significant expression changes between experimental conditions, accounting for small RNA-specific normalization.

Approach: Create a DESeqDataSet from miRNA counts, filter low-expressed miRNAs using a lower threshold than mRNA (10 reads total), run the DESeq2 pipeline, and extract results with BH-corrected p-values.

# Create DESeq2 dataset
dds <- DESeqDataSetFromMatrix(
    countData = round(counts),  # DESeq2 requires integers
    colData = coldata,
    design = ~ condition
)

# Filter low-expressed miRNAs
# miRNAs typically have fewer total counts than mRNAs
# Keep miRNAs with at least 10 reads across samples
keep <- rowSums(counts(dds)) >= 10
dds <- dds[keep, ]

# Run DESeq2
dds <- DESeq(dds)

# Get results
res <- results(dds, contrast = c('condition', 'treated', 'control'))
res <- res[order(res$padj), ]

Apply Shrinkage for Effect Sizes

# apeglm shrinkage for more accurate log2 fold changes
# Particularly important for low-count miRNAs
library(apeglm)

res_shrunk <- lfcShrink(
    dds,
    coef = 'condition_treated_vs_control',
    type = 'apeglm'
)

Filter Significant miRNAs

# Standard thresholds for miRNA DE
# padj < 0.05: FDR-corrected significance
# |log2FC| > 1: 2-fold change minimum
sig <- subset(res_shrunk, padj < 0.05 & abs(log2FoldChange) > 1)
sig <- sig[order(sig$padj), ]

# Separate up and down-regulated
up <- subset(sig, log2FoldChange > 0)
down <- subset(sig, log2FoldChange < 0)

cat('Upregulated:', nrow(up), '\n')
cat('Downregulated:', nrow(down), '\n')

edgeR Alternative

library(edgeR)

# Create DGEList
dge <- DGEList(counts = counts, group = coldata$condition)

# Filter low expression
keep <- filterByExpr(dge)
dge <- dge[keep, , keep.lib.sizes = FALSE]

# Normalize
dge <- calcNormFactors(dge)

# Design matrix
design <- model.matrix(~ condition, data = coldata)

# Estimate dispersion
dge <- estimateDisp(dge, design)

# Fit model and test
fit <- glmQLFit(dge, design)
qlf <- glmQLFTest(fit, coef = 2)

# Get results
res_edger <- topTags(qlf, n = Inf)$table

Visualization

library(ggplot2)
library(EnhancedVolcano)

# Volcano plot
EnhancedVolcano(
    res_shrunk,
    lab = rownames(res_shrunk),
    x = 'log2FoldChange',
    y = 'padj',
    pCutoff = 0.05,
    FCcutoff = 1,
    title = 'Differential miRNA Expression'
)

# MA plot
plotMA(res_shrunk, ylim = c(-4, 4))

Heatmap of DE miRNAs

library(pheatmap)

# Get normalized counts
vsd <- vst(dds, blind = FALSE)

# Select significant miRNAs
sig_mirnas <- rownames(sig)
mat <- assay(vsd)[sig_mirnas, ]

# Z-score scale rows
mat_scaled <- t(scale(t(mat)))

pheatmap(
    mat_scaled,
    annotation_col = coldata['condition'],
    cluster_rows = TRUE,
    cluster_cols = TRUE,
    show_rownames = nrow(mat) < 50
)

Export Results

# Full results with normalized counts
res_df <- as.data.frame(res_shrunk)
res_df$miRNA <- rownames(res_df)
res_df$baseMean_norm <- rowMeans(counts(dds, normalized = TRUE)[rownames(res_df), ])

write.csv(res_df, 'DE_miRNAs_full.csv', row.names = FALSE)

# Significant only
write.csv(as.data.frame(sig), 'DE_miRNAs_significant.csv')

Related Skills

• mirge3-analysis - Get miRNA counts
• mirdeep2-analysis - Alternative quantification
• target-prediction - Predict targets of DE miRNAs
• differential-expression/deseq2-basics - General DE analysis concepts