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Functional Annotation with EggNOG Mapper

Introduction

EggNOG-mapper annotates novel sequences (genes or proteins) using precomputed eggNOG orthology assignments. Typical uses include annotating genomes, transcriptomes, and metagenomic gene catalogs. Orthology-based transfer is often more specific than simple homology, because annotations are not taken from paralogs that may have diverged in function.

Methodology and the underlying database are described on the eggNOG methods page.

Open the wizard from:

  • Functional Analysis → Functional Annotation with EggNOG Mapper
  • Metagenomics → Annotation Tools → Functional Annotation with EggNOG Mapper

When you start the job from a loaded OmicsBox project, the Input page is skipped and sequences come from the project. When you start from the menu without a project, select input files on the Input page first.

Input

The Input page is shown only when no project is loaded.

  • Genes or Proteins: One or more multi-FASTA files containing gene or protein sequences (extensions such as .fasta, .faa, .fna, .fa, .fn, .ffn).

  • Input Type: How sequences are searched against eggNOG (default CDS):

  • Proteins: Amino acid sequences are searched with BLASTp.

  • CDS: Coding sequences are translated to proteins, then searched with BLASTp.
  • Genes/Contigs: Nucleotide sequences are searched in all six reading frames with BLASTx.

OmicsBox can detect whether files contain protein or nucleotide sequences and warns if the chosen Input Type does not match. Sequences longer than 2.5 Mb are filtered automatically and skipped.

Configuration

The Configuration page sets search and annotation options (Figure 1).

Search filters

  • Minimum Hit e-Value: Minimum e-value for seed ortholog hits in the homology search phase (default 1.0E-3). Lower values are more stringent; higher values are more permissive.

Annotation options

  • Taxonomic Scope: Restrict annotation to orthologs from a chosen clade. Default Adjust Automatically picks the best scope per query sequence. Select a specific taxon when you know the origin of your data.

  • Target Orthologs: Which ortholog relationships are used to transfer function (default All):

  • All: All ortholog types (recommended).

  • One to One: Single ortholog per pair of species (most stringent).
  • One to Many: One-to-one and one-to-many orthologs.
  • Many to One: One-to-one and many-to-one orthologs.

  • Many to Many: All types including many-to-many (least stringent, most comprehensive).

  • GO Evidence: Which Gene Ontology (GO) terms are transferred (default Non-Electronic):

  • Experimental: Only terms with experimental evidence codes (fewer, high-confidence terms).

  • Non-Electronic: All evidence types except electronic inference only (recommended balance of quality and coverage).

Figure 1. EggNOG Mapper wizard, Configuration page.

Results

The result table lists annotations transferred by EggNOG Mapper. You can sort, filter, and open the context menu for more actions (Figure 2). The run also produces a Summary Report with totals for GO terms, COG categories, and orthologous groups.

Figure 2. EggNOG Mapper results table.

Right-click an annotated sequence and choose Show Annotation Details to open link-outs and detailed GO information (Figure 3).

Figure 3. EggNOG Mapper annotation details.

References

Huerta-Cepas J et al. (2019). eggNOG 5.0: a hierarchical, functionally and phylogenetically annotated orthology resource based on 5090 organisms and 2502 viruses. Nucleic Acids Research, 47(D1), D309-D314.