featureCounts Tutorial: Gene Expression Quantification for RNA-Seq
📖 RNA-Seq Data Analysis Workflow — check it out for an overview.
Introduction
In RNA-seq analysis using next-generation sequencing, you obtain raw data known as FASTQ files (reads). After mapping each read to a reference genome, gene expression levels are quantified by counting the number of reads mapped to each gene.
This page walks you through how to use featureCounts, a tool for counting reads mapped to genomic features.
Installing featureCounts
The easiest way to install featureCounts is through Bioconda. Since featureCounts is part of the Subread package, you need to install Subread.
Try displaying the help message to confirm the installation.
If you see output similar to the following, the installation was successful.
Running Read Counting
Use the following command to count reads. In this example, we process four samples: sample1, sample2, sample3, and sample4.
Option Details
| Option | Description |
| -p | Counts fragments instead of individual reads. Use this option for paired-end data. |
| -t | Specifies the feature type in the GTF file to use for read counting. The default is 'exon'. |
| -g | Specifies the GTF attribute to use as the grouping unit for read counting. The default is 'gene_id'. |
In this example, reads are counted as fragments rather than individual reads, only reads mapped to exons are included, and the counts are aggregated by gene_id.
Results
Running the command above produces output like the following.
The first line contains the featureCounts version and the command that was executed. From the seventh column onward, the read count results for each sample are shown.
Columns 1 through 6 contain the following information.
| Column Number | Column Name | Description |
| 1 | Geneid | Gene ID |
| 2 | Chr | Chromosome |
| 3 | Start | Start positions of all exons, separated by semicolons. |
| 4 | End | End positions of all exons, separated by semicolons. |
| 5 | Strand | Strand orientation of all exons, separated by semicolons. |
| 6 | Length | Total gene length. If exons overlap, this value will be shorter than the sum of all individual exon lengths. |
RNA-Seq Data Analysis Software
This is an RNA-Seq Data Analysis Software recommended for those who:
✔︎ Seeking to avoid outsourcing or collaboration for RNA-Seq data analysis.
✔︎ Lacking time to learn RNA-Seq data analysis.
✔︎ Frustrated by the complexity of existing tools.
Users can perform gene expression quantification, identification of differentially expressed genes, gene ontology(GO) analysis, pathway analysis, as well as drawing volcano plots, MA plots, and heatmaps.
About the Author
BxINFO LLC
A research support company specializing in bioinformatics.
We provide tools and information to support life science research, with a focus on RNA-Seq analysis.