Overview of the FREEDA pipeline. The schematic shows the main steps, with more details in Materials and methods. Launching the FREEDA application opens a graphical user interface (gray), which prompts selection of a reference species and a gene name. First, operating on the selected reference species (blue path), FREEDA downloads the genome and uses it to curate input for the gene of interest (protein sequence, exon sequences, coding sequence, and gene sequence). Second, operating on closely related species (orange path), FREEDA downloads non-annotated genomes, searches for putative orthologous loci, retrieves these loci, finds syntenic (homologous to the reference locus) exons, and assembles coding sequences of orthologous genes based on the intron–exon boundaries known for the reference gene. Third, operating on the multiple coding sequences (brown path), FREEDA makes and curates a multiple sequence alignment, generates a phylogenetic gene tree, and detects statistical signatures of positive selection using established models measuring the rate ratio of non-synonymous to synonymous substitutions. Fourth, FREEDA maps sites with the highest probability of positive selection onto both the reference coding sequence and the structure prediction of the reference protein.