5914–5923 Nucleic Acids Research, 2005, Vol. 33, No. 18 doi:10.1093/nar/gki890 Experimental comparison and cross-validation of the Affymetrix and Illumina gene expression analysis platforms Michael Barnes, Johannes Freudenberg1, Susan Thompson, Bruce Aronow1 and Paul Pavlidis2,* Cincinnati Children’s Hospital Medical Center, Division of Rheumatology, Cincinnati OH, USA, 1Cincinnati Children’s Hospital Medical Center, Division of Biomedical Informatics, Cincinnati OH, USA and 2Columbia University Department of Biomedical Informatics, New York, NY, USA Received May 31, 2005; Revised August 11, 2005; Accepted September 25, 2005 ABSTRACT The growth in popularity of RNA expression micro- arrays has been accompanied by concerns about the reliability of the data especially when comparing between different platforms. Here, we present an evaluation of the reproducibility of microarray results using two platforms, Affymetrix GeneChips and Illumina BeadArrays. The study design is based on a dilution series of two human tissues (blood and placenta), tested in duplicate on each platform. The results of a comparison between the platforms indi- cate very high agreement, particularly for genes which are predicted to be differentially expressed between the two tissues. Agreement was strongly correlated with the level of expression of a gene. Concordance was also improved when probes on the two platforms could be identified as being likely to target the same set of transcripts of a given gene. These results shed light on the causes or failures of agreement across microarray platforms. The set of probes we found to be most highly reproducible can be used by others to help increase confidence in analyses of other data sets using these platforms. INTRODUCTION The success of gene expression microarray technology has led to the production of multiple array platforms differing in the kind of probes used (short-oligonucleotide, long- oligonucleotide, cDNA, etc.), the hybridization paradigm (competitive versus non-competitive), the labeling method and the production method (in situ polymerization, spotting, microbeads, etc.). The diversity of microarray platforms has made it challenging to compare data sets generated in different laboratories, hindering multi-institutional collaborations and reducing the usefulness of existing experimental data. When comparing gene expression studies, we not only have to consider the interesting biological factors but a plethora of technical factors including diverse sample handling, target preparation and data processing methods, as well as micro- array platform choice. In order for the beneﬁts of comparisons between two laboratories to be realized, it is crucial to under- stand the beneﬁts and limitations of each platform used as well as the cross-platform comparability. At this writing, most published gene expression studies use Affymetrix GeneChips, spotted cDNA arrays or spotted long-oligonucleotide arrays. However, new approaches are still evolving, such as the recently introduced long- oligonucleotides bead-based array by Illumina, Inc. (1). Arrays produced by Affymetrix are fabricated by in situ syn- thesis of 25mer oligonucleotides (2) while the Illumina pro- cess involves using standard oligonucleotide synthesis methods as is used for spotted long-oligonucleotides arrays. However, on Illumina arrays the oligonucleotides are attached to microbeads which are then put onto microarrays using a random self-assembly mechanism (1). In addition to the differ- ence in oligonucleotide physical attachment, the two platforms are also very different in probe selection and design procedure. Affymetrix uses multiple probes for each gene along with one-base mismatch probes intended as controls for non- speciﬁc hybridization. In contrast, the randomly generated Illumina arrays yield on the order of 30 copies of the same oligonucleotide on the array, which provide an internal tech- nical replication that Affymetrix lacks. In addition, the Affy- metrix arrays are constructed in a speciﬁc layout, with each probe synthesized at a predeﬁned location (2), while individual Illumina arrays must undergo a ‘decoding’ step in which the *To whom correspondence should be addressed. Tel: +1 212 851 5141; Fax: +1 212 851 5290; Email: pavlidis@dbmi.columbia.edu The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. 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