Quantile normalization (QN) is a technique for microarray data processing and is the default normalization method in the Robust Multi-array Average (RMA) procedure, which was primarily designed for analysing gene expression data from Affymetrix arrays. Given the abundance of Affymetrix microarrays and the popularity of the RMA method, it is crucially important that the normalization procedure is applied appropriately. In this study we carried out simulation experiments and also analysed real microarray data to investigate the suitability of RMA when it is applied to dataset with different groups of biological samples. From our experiments, we showed that RMA with QN does not preserve the biological signal included in each group, but rather it would mix the signals between the groups. We also showed that the Median Polish method in the summarization step of RMA has similar mixing effect. RMA is one of the most widely used methods in microarray data processing and has been applied to a vast volume of data in biomedical research. The problematic behaviour of this method suggests that previous studies employing RMA could have been misadvised or adversely affected. Therefore we think it is crucially important that the research community recognizes the issue and starts to address it. The two core elements of the RMA method, quantile normalization and Median Polish, both have the undesirable effects of mixing biological signals between different sample groups, which can be detrimental to drawing valid biological conclusions and to any subsequent analyses. Based on the evidence presented here and that in the literature, we recommend exercising caution when using RMA as a method of processing microarray gene expression data, particularly in situations where there are likely to be unknown subgroups of samples.

UNLABELLED: This article describes DP-Bind, a web server for predicting DNA-binding sites in a DNA-binding protein from its amino acid sequence. The web server implements three machine learning methods: support vector machine, kernel logistic regression and penalized logistic regression. Prediction can be performed using either the input sequence alone or an automatically generated profile of evolutionary conservation of the input sequence in the form of PSI-BLAST position-specific scoring matrix (PSSM). PSSM-based kernel logistic regression achieves the accuracy of 77.2%, sensitivity of 76.4% and specificity of 76.6%. The outputs of all three individual methods are combined into a consensus prediction to help identify positions predicted with high level of confidence. AVAILABILITY: Freely available at http://lcg.rit.albany.edu/dp-bind. SUPPLEMENTARY INFORMATION: http://lcg.rit.albany.edu/dp-bind/dpbind_supplement.html.

MOTIVATION: Most biological sequences contain compositionally biased segments in which one or more residue types are significantly overrepresented. The function and evolution of these segments are poorly understood. Usually, all types of compositionally biased segments are masked and ignored during sequence analysis. However, it has been shown for a number of proteins that biased segments that contain amino acids with similar chemical properties are involved in a variety of molecular functions and human diseases. A detailed large-scale analysis of the functional implications and evolutionary conservation of different compositionally biased segments requires a sensitive method capable of detecting user-specified types of compositional bias. RESULTS: We present BIAS, a novel sensitive method for the detection of compositionally biased segments composed of a user-specified set of residue types. BIAS uses the discrete scan statistics that provides a highly accurate correction for multiple tests to compute analytical estimates of the significance of each compositionally biased segment. The method can take into account global compositional bias when computing analytical estimates of the significance of local clusters. BIAS is benchmarked against SEG, SAPS and CAST programs. We also use BIAS to show that groups of proteins with the same biological function are significantly associated with particular types of compositionally biased segments.