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  • Review Article
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Use and misuse of the gene ontology annotations

Key Points

  • The Gene Ontology (GO) has a structure that allows powerful comparisons and inferences about gene functions, but its structure is often misunderstood or ignored in practice.

  • Evidence codes, annotations for unknown functions and annotation qualifiers are vital aspects of GO annotations, but these crucial features of GO annotation are often overlooked.

  • Functional profiling using GO annotations is often performed in an incorrect or inappropriate way. Important issues related to this include a tendency to perform only enrichment testing, using an incorrect reference set, lack of or an inappropriate correction for multiple comparisons, indiscriminate propagation of annotations through the hierarchy, and ignoring the correlations between GO terms.

  • Any analysis using GO annotations should cite data sources, including the version of ontology, date of annotation files, numbers and types of annotations used, and the versions and parameters of software, to ensure that results are fully reproducible.

  • Pie charts are not appropriate for displaying GO functional categorization because of the GO structure and annotation practices. Functional characterization studies should indicate the number of genes that are not mapped to any slim term, are mapped directly to the root node, or are unannotated.


The Gene Ontology (GO) project is a collaboration among model organism databases to describe gene products from all organisms using a consistent and computable language. GO produces sets of explicitly defined, structured vocabularies that describe biological processes, molecular functions and cellular components of gene products in both a computer- and human-readable manner. Here we describe key aspects of GO, which, when overlooked, can cause erroneous results, and address how these pitfalls can be avoided.

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Figure 1: Simple trees versus directed acyclic graphs.
Figure 2: Using gene ontology (GO) to bin the yeast genome into broad biological process categories.

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We are grateful to the GO Consortium for their efforts in developing, maintaining and making accessible the GO ontology and annotations. We thank S. Carbon and C. Mungall for their help with SQL queries to the GO database and the following individuals for feedback on this manuscript: M. Ashburner, E. Camon, P. D'Eustachio, E. Dimmer, P. Gaudet, R. Huntley, R. Lovering, C. Mungall, S. Twigger, and K. Van Auken.

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Correspondence to Seung Yon Rhee or Sorin Draghici.

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Related links

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Seung Yon Rhee's hompage

Sorin Draghici's homepage

An Introduction to the Gene Ontology

Gene Ontology (GO) project

GO annotation conventions

GO annotation project at the European Bioinformatics Institute (GOA)

GO downloads

GO Slim and Subset Guide

Interpro database

ISI Web of Knowledge


Princeton University's GO Term Mapper

Reference genome annotation project at GO

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Yon Rhee, S., Wood, V., Dolinski, K. et al. Use and misuse of the gene ontology annotations. Nat Rev Genet 9, 509–515 (2008).

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