The article introduces eggNOG-mapper v2, an upgraded version of a tool for functional annotation based on precomputed orthology assignments, optimized for large-scale (meta)genomic data sets. Key improvements include: 1. **Full Update of Databases**: The tool now uses eggNOG v5, covering 4.4 million ortholog groups and more than twice the number of organisms compared to the previous version. 2. **Efficiency Enhancements**: eggNOG-mapper v2 offers faster query processing and parallelization, reducing the impact of slow I/O disk operations. 3. **New Features**: - **De novo Gene Prediction**: Direct prediction of open reading frames (ORFs) from raw contigs using Prodigal. - **Built-in Pairwise Orthology Prediction**: Fast protein domain discovery and automated GFF decoration. - **Custom Taxonomic Scopes**: Users can create custom annotation databases for specific taxonomic groups. - **Orthology Reports**: Generation of pairwise orthology relationships for each query against eggNOG v5 organisms. - **Annotation Sources**: Integrated reports with various functional annotations, including KEGG pathways, Gene Ontology, EC numbers, and more. - **Protein Domain Annotations**: PFAM and SMART domain predictions, with options for de novo annotation. The tool is available as a standalone application and an online service, providing a more efficient, versatile, and scalable automated functional annotation workflow. The article also compares eggNOG-mapper v2 with other functional annotation tools, highlighting its strengths in metagenomic scale annotation and custom taxonomic scope adjustments.The article introduces eggNOG-mapper v2, an upgraded version of a tool for functional annotation based on precomputed orthology assignments, optimized for large-scale (meta)genomic data sets. Key improvements include: 1. **Full Update of Databases**: The tool now uses eggNOG v5, covering 4.4 million ortholog groups and more than twice the number of organisms compared to the previous version. 2. **Efficiency Enhancements**: eggNOG-mapper v2 offers faster query processing and parallelization, reducing the impact of slow I/O disk operations. 3. **New Features**: - **De novo Gene Prediction**: Direct prediction of open reading frames (ORFs) from raw contigs using Prodigal. - **Built-in Pairwise Orthology Prediction**: Fast protein domain discovery and automated GFF decoration. - **Custom Taxonomic Scopes**: Users can create custom annotation databases for specific taxonomic groups. - **Orthology Reports**: Generation of pairwise orthology relationships for each query against eggNOG v5 organisms. - **Annotation Sources**: Integrated reports with various functional annotations, including KEGG pathways, Gene Ontology, EC numbers, and more. - **Protein Domain Annotations**: PFAM and SMART domain predictions, with options for de novo annotation. The tool is available as a standalone application and an online service, providing a more efficient, versatile, and scalable automated functional annotation workflow. The article also compares eggNOG-mapper v2 with other functional annotation tools, highlighting its strengths in metagenomic scale annotation and custom taxonomic scope adjustments.