Open Access Highly Accessed Methodology article

Unsupervised automated high throughput phenotyping of RNAi time-lapse movies

Henrik Failmezger124, Holger Fröhlich3 and Achim Tresch12*

Author Affiliations

1 Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, 50829, Cologne, Germany

2 Institute for Genetics, University of Cologne, Zuelpicher Str. 47a, 50674, Cologne, Germany

3 Algorithmic Bioinformatics, Bonn-Aachen International Center for IT, Rheinische, Friedrich-Wilhelms-Universität, Bonn, Dahlmannstr. 2, 53113, Bonn, Germany

4 Gene Center and Department of Biochemistry, Center for Integrated Protein Science CIPSM, Ludwigs-Maximilian University, Munich 81377, Germany

For all author emails, please log on.

BMC Bioinformatics 2013, 14:292  doi:10.1186/1471-2105-14-292

Published: 4 October 2013



Gene perturbation experiments in combination with fluorescence time-lapse cell imaging are a powerful tool in reverse genetics. High content applications require tools for the automated processing of the large amounts of data. These tools include in general several image processing steps, the extraction of morphological descriptors, and the grouping of cells into phenotype classes according to their descriptors. This phenotyping can be applied in a supervised or an unsupervised manner. Unsupervised methods are suitable for the discovery of formerly unknown phenotypes, which are expected to occur in high-throughput RNAi time-lapse screens.


We developed an unsupervised phenotyping approach based on Hidden Markov Models (HMMs) with multivariate Gaussian emissions for the detection of knockdown-specific phenotypes in RNAi time-lapse movies. The automated detection of abnormal cell morphologies allows us to assign a phenotypic fingerprint to each gene knockdown. By applying our method to the Mitocheck database, we show that a phenotypic fingerprint is indicative of a gene’s function.


Our fully unsupervised HMM-based phenotyping is able to automatically identify cell morphologies that are specific for a certain knockdown. Beyond the identification of genes whose knockdown affects cell morphology, phenotypic fingerprints can be used to find modules of functionally related genes.