Agent skill

bio-rna-quantification-featurecounts-counting

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Install this agent skill to your Project

npx add-skill https://github.com/FreedomIntelligence/OpenClaw-Medical-Skills/tree/main/skills/bio-rna-quantification-featurecounts-counting

SKILL.md

name: bio-rna-quantification-featurecounts-counting description: Count reads per gene from aligned BAM files using Subread featureCounts. Use when processing BAM files from STAR/HISAT2 to generate gene-level counts for DESeq2/edgeR. tool_type: cli primary_tool: featureCounts measurable_outcome: Execute skill workflow successfully with valid output within 15 minutes. allowed-tools:

  • read_file
  • run_shell_command

featureCounts Counting

Count reads mapping to genomic features (genes, exons) from BAM files.

Basic Usage

bash
# 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

bash
# 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

bash
# 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

bash
# 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

bash
# 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

bash
# 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

bash
# 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
  1. counts.txt.summary - Assignment statistics
Status                  sample1.bam  sample2.bam
Assigned                1523456      1678234
Unassigned_Unmapped     12345        11234
Unassigned_NoFeatures   234567       245678

Extract Count Matrix

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

Python Processing

python
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

r
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

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