featureCounts

FeatureCounts is a program in the Subread software package designed to summarize mapped sequencing reads into counts for predefined genomic features. It is widely used for gene- and exon-level quantification in RNA-Seq and can be applied to other sequencing assays. The input consists of alignment files in BAM or SAM format and an annotation file in GTF or GFF format. Users specify the feature type to count (for example exons or genes) and the attribute to group features (such as gene_id). The program supports strand-specific counting, paired-end data, and counting of reads that overlap multiple features or map to multiple genomic locations, depending on options chosen. It also offers the ability to count reads at different feature levels and to produce counts suitable for downstream differential expression analysis.

Output is a tab-delimited file containing counts per feature per sample, along with optional summary statistics. FeatureCounts can be used directly from the command line and via the R interface provided in the Rsubread package, enabling integration with R-based pipelines and tools such as DESeq2, edgeR, and limma.

Strengths include speed, memory efficiency, and flexibility in feature definition and counting strategies. Limitations stem from dependence on annotation quality and the need to select appropriate counting parameters for specific experimental designs. FeatureCounts was introduced as part of the Subread package and is described in the literature by Liao, Smyth, and Shi (2014).