featureCounts — RNA-seq Read Counting

featureCounts — RNA-seq Read Counting

Overview

featureCounts (part of the Subread package) assigns sequencing reads in BAM files to genomic features defined in a GTF/GFF annotation. It counts how many reads overlap each gene (or exon, intron, or custom feature), producing a gene × sample count matrix suitable for differential expression analysis with DESeq2 or edgeR. featureCounts processes multiple BAM files in a single command, reporting read assignment statistics (assigned, unassigned by category) alongside the count matrix. It is the standard counting step after STAR alignment in RNA-seq pipelines.

When to Use

• Generating gene-level count matrices from STAR-aligned BAM files for DESeq2 or edgeR
• Counting reads from multiple samples simultaneously in a single featureCounts command
• Handling stranded RNA-seq libraries where sense/antisense assignment matters
• Producing exon-level or custom-feature counts (e.g., for splicing analysis with DEXSeq)
• Verifying strandedness of an RNA-seq library when protocol documentation is unavailable
• Use Salmon instead when no BAM file exists and fast pseudoalignment is preferred
• Use HTSeq-count as an alternative with slower but more flexible counting modes

Prerequisites

• Software: Subread package (contains featureCounts)
• Input: Sorted BAM files from STAR or HISAT2, plus a matching GTF annotation file

Check before installing: The tool may already be available in the current environment (e.g., inside a pixi / conda env). Run command -v featureCounts first and skip the install commands below if it returns a path. When running inside a pixi project, invoke the tool via pixi run featureCounts rather than bare featureCounts.

# Install with conda (recommended)
conda install -c bioconda subread

# Verify
featureCounts -v
# featureCounts v2.0.6

# Alternative: install via apt (Ubuntu/Debian)
sudo apt-get install subread

Pre-flight Interview

Settle these with the user before writing any analysis code.