Large-scale gene function analysis with the PANTHER classification system


The PANTHER (protein annotation through evolutionary relationship) classification system ( is a comprehensive system that combines gene function, ontology, pathways and statistical analysis tools that enable biologists to analyze large-scale, genome-wide data from sequencing, proteomics or gene expression experiments. The system is built with 82 complete genomes organized into gene families and subfamilies, and their evolutionary relationships are captured in phylogenetic trees, multiple sequence alignments and statistical models (hidden Markov models or HMMs). Genes are classified according to their function in several different ways: families and subfamilies are annotated with ontology terms (Gene Ontology (GO) and PANTHER protein class), and sequences are assigned to PANTHER pathways. The PANTHER website includes a suite of tools that enable users to browse and query gene functions, and to analyze large-scale experimental data with a number of statistical tests. It is widely used by bench scientists, bioinformaticians, computer scientists and systems biologists. In the 2013 release of PANTHER (v.8.0), in addition to an update of the data content, we redesigned the website interface to improve both user experience and the system's analytical capability. This protocol provides a detailed description of how to analyze genome-wide experimental data with the PANTHER classification system.

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Figure 1: Overview of PANTHER infrastructure.
Figure 2: Sample phylogenetic tree from PANTHER.
Figure 3: Example of a PANTHER pathway diagram.
Figure 4
Figure 7
Figure 8: Summary of the results from the statistical overrepresentation test displayed in a table.
Figure 9: Results from the statistical enrichment test.
Figure 10: Graph view of results from the statistical enrichment test.
Figure 11: Results from the statistical enrichment test as visualized in the pathway diagram.
Figure 5: Results of functional classification displayed as a gene list page.
Figure 6: PANTHER pie chart results using Supplementary Data 1 as the input gene list file.
Figure 12: Results of the statistical overrepresentation test viewed in the PANTHER pathway, 'Heterotrimeric G protein signaling pathway – Gi-α and Gs-α–mediated pathway (PANTHER accession code P00026)'.


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We thank the Reference Proteome team, especially C. McAnulla and M. Martin, for their support in providing up-to-date Reference Proteome data set, and we thank Y. Matsuoka and K. Manami from the Systems Biology Institute Japan for their support on CellDesigner and pathway file update. This work is supported by the US National Institutes of Health (NIH)/National Institute of General Medical Sciences (NIGMS) grant no. GM081084 to P.D.T. Funding for open access was provided by the University of Southern California.

Author information




A.M. developed the software code for the website. J.T.C. maintained the database and web servers. H.M. generated the content of the system and supervised the project. P.D.T. provided the funding and supervised the project. H.M. wrote the manuscript with contributions from all the authors.

Corresponding author

Correspondence to Huaiyu Mi.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Data 1

File containing 555 gene IDs, 500 of which can be classified in PANTHER. It can be used as a sample upload file to test the functional classification tools and the overrepresentation tool. (TXT 10 kb)

Supplementary Data 2

File containing 19,911 genes, with IDs in the first column and numeric experimental values in the second column. This file can be used as a sample upload file for all tools. (TXT 381 kb)

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Mi, H., Muruganujan, A., Casagrande, J. et al. Large-scale gene function analysis with the PANTHER classification system. Nat Protoc 8, 1551–1566 (2013).

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