Open Access Research article

A verification protocol for the probe sequences of Affymetrix genome arrays reveals high probe accuracy for studies in mouse, human and rat

Rudi Alberts1*, Peter Terpstra2, Menno Hardonk2, Leonid V Bystrykh3, Gerald de Haan3, Rainer Breitling1, Jan-Peter Nap14 and Ritsert C Jansen12

Author Affiliations

1 Groningen Bioinformatics Centre, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751 NN Haren, The Netherlands

2 Groningen Bioinformatics Centre, University Medical Centre Groningen, University of Groningen, 9713 AV Groningen, The Netherlands

3 Department of Cell Biology, section Stem Cell Biology, University Medical Centre Groningen, University of Groningen, 9713 AV Groningen, The Netherlands

4 Bioinformatics Expertise Center, Institute for Life Science & Technology, Hanze University Groningen, 9747 AS Groningen, The Netherlands

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BMC Bioinformatics 2007, 8:132  doi:10.1186/1471-2105-8-132

Published: 20 April 2007



The Affymetrix GeneChip technology uses multiple probes per gene to measure its expression level. Individual probe signals can vary widely, which hampers proper interpretation. This variation can be caused by probes that do not properly match their target gene or that match multiple genes. To determine the accuracy of Affymetrix arrays, we developed an extensive verification protocol, for mouse arrays incorporating the NCBI RefSeq, NCBI UniGene Unique, NIA Mouse Gene Index, and UCSC mouse genome databases.


Applying this protocol to Affymetrix Mouse Genome arrays (the earlier U74Av2 and the newer 430 2.0 array), the number of sequence-verified probes with perfect matches was no less than 85% and 95%, respectively; and for 74% and 85% of the probe sets all probes were sequence verified. The latter percentages increased to 80% and 94% after discarding one or two unverifiable probes per probe set, and even further to 84% and 97% when, in addition, allowing for one or two mismatches between probe and target gene. Similar results were obtained for other mouse arrays, as well as for human and rat arrays. Based on these data, refined chip definition files for all arrays are provided online. Researchers can choose the version appropriate for their study to (re)analyze expression data.


The accuracy of Affymetrix probe sequences is higher than previously reported, particularly on newer arrays. Yet, refined probe set definitions have clear effects on the detection of differentially expressed genes. We demonstrate that the interpretation of the results of Affymetrix arrays is improved when the new chip definition files are used.