Evaluation of normalization methods for cDNA microarray data by k-NN classification

Background: Non-biological factors give rise to unwanted variations in cDNA microarray data. There are many normalization methods designed to remove such variations. However, to date there have been few published systematic evaluations of these techniques for removing variations arising from dye biases in the context of downstream, higher-order analytical tasks such as classification. Results: Ten location normalization methods that adjust spatialand/or intensity-dependent dye biases, and three scale methods that adjust scale differences were applied, individually and in combination, to five distinct, published, cancer biology-related cDNA microarray data sets. Leave-one-out cross-validation (LOOCV) classification error was employed as the quantitative end-point for assessing the effectiveness of a normalization method. In particular, a known classifier, k-nearest neighbor (k-NN), was estimated from data normalized using a given technique, and the LOOCV error rate of the ensuing model was computed. We found that k-NN classifiers are sensitive to dye biases in the data. Using NONRM and GMEDIAN as baseline methods, our results show that single-bias-removal techniques which remove either spatialdependent dye bias (referred later as spatial effect) or intensity-dependent dye bias (referred later as intensity effect) moderately reduce LOOCV classification errors; whereas double-bias-removal techniques which remove both spatialand intensity effect reduce LOOCV classification errors even further. Of the 41 different strategies examined, three two-step processes, IGLOESS-SLFILTERW7, ISTSPLINE-SLLOESS and IGLOESS-SLLOESS, all of which removed intensity effect globally and spatial effect locally, appear to reduce LOOCV classification errors most consistently and effectively across all data sets. We also found that the investigated scale normalization methods do not reduce LOOCV classification error. Conclusion: Using LOOCV error of k-NNs as the evaluation criterion, three double-bias-removal normalization strategies, IGLOESS-SLFILTERW7, ISTSPLINE-SLLOESS and IGLOESS-SLLOESS, outperform other strategies for removing spatial effect, intensity effect and scale differences from cDNA microarray data. The apparent sensitivity of k-NN LOOCV classification error to dye biases suggests that this criterion provides an informative measure for evaluating normalization methods. All the computational tools used in this study were implemented using the R language for statistical computing and graphics. Published: 26 July 2005 BMC Bioinformatics 2005, 6:191 doi:10.1186/1471-2105-6-191 Received: 17 December 2004 Accepted: 26 July 2005 This article is available from: http://www.biomedcentral. com/1471-2105/6/191 © 2005 Wu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Page 1 of 21 (page number not for citation purposes) BMC Bioinformatics 2005, 6:191 http://www.biomedcentral. com/1471-2105/6/191 Background Molecular profiling technology allows for the simultaneous assaying of the abundance of tens of thousands of transcripts in a biological sample. Once these abundance values have been obtained for many samples, prevalent higher-order data analyses may include clustering, classification, feature selection, and network estimation. A variety of algorithms seeking to address these higher-order tasks have been investigated and applied, to interpret gene expression patterns and to generate biological predictions. However, the accuracy of these predictions may depend on the low-level transformations utilized to produce abundance values from raw measurements, i.e., data pre-processing may be a critical factor in determining the validity and success of downstream studies. Some key preprocessing steps for profiling data include image quantification and normalization. Several image analysis software (e.g., GenePix and SPOT) have been designed for image analysis of the spots on microarrays [1,2]. Background estimation has also been considered as an important issue in image quantification, however, evidence [2,3] showed that 'inappropriate' local background adjustment could add noise into the microarray data and thus be detrimental to the downstream studies. Background adjustment, therefore, is still an issue to be resolved. After image analysis, normalization usually needs to be performed. It is a procedure designed to minimize the unwanted variations in measurements arising from the technology, but to retain the intrinsic biological variations, and is also the focus of this work. In this study, we examined normalization in the context of a particular transcriptional profiling platform, cDNA microarrays [4-6], and the specific analytical task of classifying biological samples characterized by gene expression profiles. In cDNA microarray-based investigations, RNA from two samples are reverse-transcribed and labeled with distinct (red and green) fluorescent dyes, then hybridized to a microarray spotted with DNA sequences ("probes"). An ensuing scanned image of the microarray is processed to yield an intensity measurement for each dye at every spot (Figure 1). If R and G are the spot-specific, quantitated, fluorescent intensities of the target and reference expression signals respectively, relative gene expression is defined as the log ratio M = log2(R / G), and average expression is the log intensity . Based on different biological assumptions and design principles, many normalization methods for cDNA microarray data have been proposed. Global normalization techniques adjust the center (e.g., mean or median) of the distribution of the log ratio M values on each microarray to a constant [1,7-9]. These methods, however, do not correct for any intensityor spatial effect. A variety of techniques have been proposed to remove intensity effect. A non-linear approach employs robust locally weighted regression (lowess) [10] to smooth the dependence of log ratios on intensities [4,11,12]. The basic assumption of this approach is either that the majority of genes are not differentially expressed, or that genes are influenced by random effects (i.e., the numbers of upregulated and down-regulated genes are similar) [4,11,12]. A 'qspline' method uses a target array to adjust R and G values so that their distribution is similar for all arrays [13], but the performance of this method may depend upon the choice of the baseline array [14]. A composite method employs both external control samples and total genes on a microarray to remove intensity effect [15]. To relax critical biological assumptions, 'housekeeping-gene'-related methods first identify non-differentiallyexpressed genes, and then use these genes for normalization [16-18]. Semi-linear models are designed to account for the effects of print-tips (PTs), signal intensity, and A RG = log2 A scanned image of an illustrative cDNA microarray Figure 1 A scanned image of an illustrative cDNA microarray. The configuration (layout of spots) can be described via a previously defined notation encompassing four numbers (ngr, ngc, nsr, nsc) [12]. A print-tip (PT) group is a set of spots arranged in a grid with "nsr" rows and "nsc" columns. A microarray is a set of PT groups arranged in a pattern of "ngr" rows and "ngc" columns. The configuration of the microarray shown is (ngr = 2, ngc = 2, nsr = 24, nsc = 24), i.e., 2 × 2 PT groups each composed of 24 × 24 spots. The terms "local" and "global" level refer to the spots in a PT group and the entire microarray respectively. Page 2 of 21 (page number not for citation purposes) BMC Bioinformatics 2005, 6:191 http://www.biomedcentral. com/1471-2105/6/191 differences in gene expression levels jointly in a single model [19,20]. The removal of intensity effect at the PT level can partially remove spatial effect [4,11]. To remove spatial effect more completely, the dependence of M values on physical position can be smoothed using lowess [12], or can be eliminated using weighted mean [13] or median filter methods [17], both of which assume that differentially expressed genes are not co-localized in the neighboring spots. Since spatialand intensity effect may be mutually dependent, a method that removes global spatial effect and global intensity effect in a single step has been proposed [21]. Whereas the above location normalization methods remove spatialand intensity effect, scale normalization methods adjust differences in the scale of M values within and/or between microarrays. The assumption is that since the majority of genes are not differentially expressed, the scale of their M values should be constant. A robust estimate of the scale factor for scale normalization is median absolute deviation [15]. Normalization approaches seek to ensure that dye effect is removed, while biological variations are retained. Spatialand intensity effect and scale effect arise from printing, hybridization, scanning, or other technical factors, and can mask the signals arising from genuine biological variations in gene expression. Visual aids used to assess the effectiveness of normalization methods [11,13,15,21] include scatter plots of log ratio (M) versus average log intensity (A) ("MA plots"). Spatial plots are a color-coded representation of each spot on a microarray that depicts M values, or a quality (e.g., shape, size) measure of some test statistic. These two types of diagnostic plots [4,21] suggest that raw M values are often biased estimates of relative expression and that the dye intensities per spot need to be adjusted. Quantitative criteria used to assess the robustness of normalization methods in removing dye effect include (i) rank variations of spot intensity in non-normalized versus normalized data [9,22], and (ii) correl