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

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.Microarrays are widely used to study genome-wide gene expression levels. A frequently used type of microarray is the Affymetrix GeneChip [1]. This technology uses multiple probes per gene (probe set) to measure the amount of mRNA present (target). For reasons of specificity, probes are chosen to be complementary to a unique part of the target sequence. Although all probes from a single probe set should measure the same amount of mRNA, the hybridization signals of individual probes for a given mRNA molecule may vary widely. This is believed to be caused by variations in molecular characteristics of the probe sequence, such as GC content and secondary structure, and corrections have been proposed to calculate true expression levels averaged over probe signals [2,3]. However, another reason for the variation in signal between probes could be misdesigned probes,