Differential Expression Analysis

Identify significantly differentially expressed genes with interactive thresholds

Volcano plots are a standard method for visualizing differential expression (DE) results. They display thousands of genes simultaneously, making it easy to identify:

• Upregulated genes - Higher expression in treatment vs. control
• Downregulated genes - Lower expression in treatment vs. control
• Statistically significant changes - Based on p-value or FDR thresholds
• Biologically meaningful changes - Based on fold-change magnitude

The Volcano module provides real-time threshold adjustment, allowing you to explore different significance cutoffs without re-analyzing your data.

Input Requirements

Required Files

Differential Expression File

• Filename must contain keywords: diff, differential, de, expression, diffexp
• Examples: DESeq2_results.csv, differential_expression_WT_vs_KO.tsv

File Format

Your DE file must contain these columns:

1. Gene/Transcript Identifier

   • Column can have any name (e.g., gene_id, symbol, ensembl_id)
   • Each row represents one gene/transcript

2. Log Fold Change

   • Column name should include: logfc, log2fc, logfoldchange
   • Examples: log2FoldChange, logFC, Log_Fold_Change
   • Positive values = upregulated, Negative values = downregulated

3. P-value or Adjusted P-value

   • Platform searches for (in priority order):
     • Adjusted: padj, fdr, qvalue, adj_p_val
     • Raw: pval, pvalue, p_value
   • Uses adjusted p-value if available, otherwise raw p-value

Gene vs Transcript Level

• If filename contains transcript, treated as transcript-level DE (In case of server side sorting)
• Otherwise, treated as gene-level DE
• Multiple DE files can be uploaded for different comparisons

Understanding the Volcano Plot

Step 1: Select Contrast and Search Genes

The top panel allows users to choose a differential-expression comparison (contrast) and optionally highlight specific genes of interest.

Contrast Selection

• Choose a comparison from the Contrast dropdown (e.g., A3).
• The volcano plot updates automatically.

Search Genes

• Use the Search Genes box to highlight specific gene IDs.
• Highlighted genes appear in orange on the plot.

Features

• Multiple genes can be selected at once.
• Hovering over highlighted points shows their identifiers.

Contrast and search bar interface

Step 2: Understand the Volcano Plot

The volcano plot helps identify significantly upregulated and downregulated genes.

Plot Layout

• X-axis (log2FC)
  • Negative values → downregulated
  • Positive values → upregulated
  • Zero → no change
• Y-axis (-log10(p-value))
  Higher values indicate greater statistical significance.

Color Coding

• 🔴 Upregulated — log2FC > threshold AND p-value < threshold
• 🔵 Downregulated — log2FC < –threshold AND p-value < threshold
• ⚫ Not significant — does not meet significance criteria
• 🟡 Highlighted genes — genes selected via Search

Visual Indicators

• Vertical dashed lines show fold change thresholds.
• Horizontal dashed line marks the significance threshold.

Volcano plot layout with thresholds

Step 3: Adjust Thresholds in Real Time

The volcano module allows real-time filtering using two threshold controls.

1. Fold Change Threshold (X)

• Controls the minimum |log2FC| required to classify a gene as up/downregulated.
• Increasing this value highlights more strongly regulated genes.

2. Significance Threshold (Y)

• Controls the minimum statistical significance required.
• Lowering the p-value threshold increases strictness.

Live Gene Counts

A summary bar shows:

• Upregulated
• Downregulated
• Not significant
• Highlighted genes
• Total genes

Step 4: Configure Plot Settings

The Settings panel allows configuration of axes and scale options.

Axis Configuration

• X-Axis Column — typically logFC
• Y-Axis Column — typically p-value or adjusted p-value

Logarithmic Scale

• Option to apply log transformation on the Y-axis.
• Zero or negative values are adjusted automatically to remain visible.

Additional Options

• Download Data — download DE files
• Preview Files — view differential expression tables
• Apply Changes — update plot with new settings

Settings panel for volcano plot