Bio Rna Quantification Featurecounts Counting

Basic Usage

# Single sample
featureCounts -a annotation.gtf -o counts.txt aligned.bam

# Multiple samples (recommended - single matrix output)
featureCounts -a annotation.gtf -o counts.txt sample1.bam sample2.bam sample3.bam

# All BAMs in directory
featureCounts -a annotation.gtf -o counts.txt *.bam

Paired-End Data

# Count fragments, not reads (required for paired-end)
featureCounts -p --countReadPairs -a annotation.gtf -o counts.txt *.bam

# Check proper pairs only
featureCounts -p --countReadPairs -B -C -a annotation.gtf -o counts.txt *.bam

Flags:

• -p - Input is paired-end
• --countReadPairs - Count fragments instead of reads
• -B - Only count properly paired reads
• -C - Don't count chimeric fragments

Strand-Specific Libraries

# Unstranded (default)
featureCounts -s 0 -a annotation.gtf -o counts.txt *.bam

# Forward stranded (e.g., dUTP, NSR)
featureCounts -s 1 -a annotation.gtf -o counts.txt *.bam

# Reverse stranded (e.g., Illumina TruSeq, most common)
featureCounts -s 2 -a annotation.gtf -o counts.txt *.bam

Determining strandedness: Use infer_experiment.py from RSeQC or check library prep protocol.

Feature Types

# Count at gene level (default)
featureCounts -t exon -g gene_id -a annotation.gtf -o counts.txt *.bam

# Count at transcript level
featureCounts -t exon -g transcript_id -a annotation.gtf -o counts.txt *.bam

# Count CDS only
featureCounts -t CDS -g gene_id -a annotation.gtf -o counts.txt *.bam

Flags:

• -t - Feature type in GTF (default: exon)
• -g - Meta-feature attribute (default: gene_id)

Multi-Mapping Reads

# Discard multi-mappers (default, recommended for DE)
featureCounts -a annotation.gtf -o counts.txt *.bam

# Count multi-mappers (fractional)
featureCounts -M --fraction -a annotation.gtf -o counts.txt *.bam

# Count multi-mappers (full count to each location)
featureCounts -M -a annotation.gtf -o counts.txt *.bam

Overlapping Features

# Discard reads overlapping multiple features (default)
featureCounts -a annotation.gtf -o counts.txt *.bam

# Count reads overlapping multiple features
featureCounts -O -a annotation.gtf -o counts.txt *.bam

# Fractional count for overlaps
featureCounts -O --fraction -a annotation.gtf -o counts.txt *.bam

Performance Options

# Use multiple threads
featureCounts -T 8 -a annotation.gtf -o counts.txt *.bam

# Use less memory (slower)
featureCounts --largeBAM -a annotation.gtf -o counts.txt *.bam

Output Files

featureCounts produces two files:

1. counts.txt - Main count matrix

Geneid  Chr     Start   End     Strand  Length  sample1.bam  sample2.bam
GENE1   chr1    100     500     +       400     1523         1891
GENE2   chr1    1000    2000    -       1000    892          756

2. counts.txt.summary - Assignment statistics

Status                  sample1.bam  sample2.bam
Assigned                1523456      1678234
Unassigned_Unmapped     12345        11234
Unassigned_NoFeatures   234567       245678

Extract Count Matrix

# Remove first 6 columns (metadata) to get just counts
cut -f1,7- counts.txt | tail -n +2 > count_matrix.txt

Python Processing

import pandas as pd

counts = pd.read_csv('counts.txt', sep='\t', comment='#')
count_matrix = counts.set_index('Geneid').iloc[:, 5:]  # Skip metadata columns
count_matrix.columns = [c.replace('.bam', '') for c in count_matrix.columns]
count_matrix.to_csv('count_matrix.csv')

R Processing

counts <- read.table('counts.txt', header=TRUE, row.names=1, skip=1)
count_matrix <- counts[, 6:ncol(counts)]  # Skip metadata columns
colnames(count_matrix) <- gsub('.bam', '', colnames(count_matrix))

Common Issues

Low assignment rate:

• Check strandedness setting (-s)
• Verify GTF matches reference genome version
• Check BAM alignment quality

Zero counts for known expressed genes:

• Ensure feature type matches GTF (-t exon vs -t gene)
• Check gene_id attribute name in GTF

Related Skills

• alignment-files/sam-bam-basics - Input BAM file handling
• genome-intervals/gtf-gff-handling - GTF annotation files
• differential-expression/deseq2-basics - Downstream analysis with counts
• rna-quantification/count-matrix-qc - QC of count data