Microarray segmentation methods significantly influence data precision.

Little consideration has been given to the effect of different segmentation methods on the variability of data derived from microarray images. Previous work has suggested that the significant source of variability from microarray image analysis is from estimation of local background. In this study, we used Analysis of Variance (ANOVA) models to investigate the effect of methods of segmentation on the precision of measurements obtained from replicate microarray experiments. We used four different methods of spot segmentation (adaptive, fixed circle, histogram and GenePix) to analyse a total number of 156 172 spots from 12 microarray experiments. Using a two-way ANOVA model and the coefficient of repeatability, we show that the method of segmentation significantly affects the precision of the microarray data. The histogram method gave the lowest variability across replicate spots compared to other methods, and had the lowest pixel-to-pixel variability within spots. This effect on precision was independent of background subtraction. We show that these findings have direct, practical implications as the variability in precision between the four methods resulted in different numbers of genes being identified as differentially expressed. Segmentation method is an important source of variability in microarray data that directly affects precision and the identification of differentially expressed genes.