In situ analysis of cross-hybridisation on microarrays and the inference of expression correlation

We demonstrate a positive relation between off-target reporter alignment strength and expression correlation in data from oligonucleotide genechips. Furthermore, we describe a method that allows the identification, from their expression data, of individual probe sets affected by off-target hybridization.The effects of off-target hybridization on expression correlation coefficients can be substantial, and can be alleviated by more accurate mapping between microarray reporters and the target transcriptome. We recommend attention to the mapping for any microarray analysis of gene expression patterns.Microarrays are a valuable tool in functional genomics research. The breadth of their applications is reflected by the myriad of computational methods that have been developed for their analysis in the last decade. One popular practice is to compare expression patterns of genes by calculating correlation coefficients on expression level estimates across a set of conditions. Many downstream analysis tools are based on the presence or absence of correlation in the expression profiles of genes, like the inference of co-expression [1-5], gene regulatory [6] and Bayesian networks [7-10] and the study of gene family evolution [11,12]. From a biological point of view, these approaches are useful and informative, but here we show that if care has not been taken as to how these correlations are calculated and how the reporters for each transcript are selected, incorrect conclusions can be drawn.A gene is represented on a microarray by one or more reporters, i. e. nucleotide sequences that are designed to uniquely match its transcript, or transcripts if different splice variants exist [13]. Affymetrix GeneChips are the most widely used microarray platform, and a wealth of data measured on these arrays is publicly available. Affymetrix reporters are 25-mer oligonucleotides whose sequence is complementary to the intended target. Each target is represented by a set of reporters, called