ࡱ> q` R:bjbjqPqPq ::2 DDDDDDDD;;;4<t<,>af=pP>P>P>P>MMM```````$bh e`D|RLM|R|R`DDP>P>[`0Z0Z0Z|RDP>DP>`0Z|R`0Z0Z\DD\P>= #/;ZWN\]a0>a\,eX6e\eD\MN0ZkOPmMMM``YRMMM>a|R|R|R|R$0"0"DDDDDD Effect of data processing on the identifying differentially expressed genes with moderate effects Seoae Cho EMBED Equation.3 , Eunjee Lee EMBED Equation.3 , Youngchul Kim EMBED Equation.3 , Eun Kyung Lee EMBED Equation.3  , Teasung Park EMBED Equation.3  1. Interdisciplinary Program in Bioinformatics, Seoul National University, Seoul, Korea 2. Department of Statistics, Seoul National University, Seoul, Korea 1. Introduction Chronic Fatigue Syndrome (CFS) is known to be a complex disease with several genes related with. In order to identify differentially expressed genes, a large gene expression data set was obtained using a one-channel microarray experiment conducted on 173 patients. The patients have been classified into five group of CFS and 20160 genes were represented. Unlike the usual microarray experiment, this study contained very large number of slides. Unfortunately, however, our preliminary analysis yielded a few significant genes differentially expressed among five groups. This result was quite surprising, because 173 would be regarded as a quite large sample size in the usual microarray experiment. Thus, we expect that the gene effect is quite moderate. Recently, microarray data interpretation was mainly focused on the comparisons between the high density oligonucleotide-based chip and two-channel cDNA microarrays [1-5]. The current existing literature offers little guidance on how to decide which method to use and how to compare different methods for their final results. These effects are most problematic, especially for one-channel microarray measurements when dealing with large grouped samples [2]. Thus, a more reliable method is required to determine an appropriate combination of individual basic processing steps for a given dataset in order to improve the validity of one channel expression data analysis. In this paper, we focus on the effect of data processing on the interpretation of gene expression data for a one-channel microarray experiment when the gene effect is not large. We expecially focus on evaluating the effect of normalization method on identifying differentially expressed genes. 2. Data Pre-Processing We analyzed a large gene expression data set obtained from a one-channel oligonucleotide experiment conducted on 173 patients that have been classified into five group of Chronic Fatigue Syndrome and 20160 genes are represented. The data in this study is about CFS that has no diagnostic clinical signs. It is unclear if it represents a single illness [6]. The samples were classified based on information from clinical consensus into five groups: 36 number of patients in Non-Fatigued (NF) group, 46 number of patients in Chronic Fatigue Syndrome (CFS) group, 47 number of patients in Chronically fatigued but not CFS because of insufficient number of symptoms (ISF) group, 20 number of patients in Chronically fatigued but with ISF and a major depressive disorder with melancholic features (ISF-MDDm) group, 19 number of patients in CFS except that a major depressive disorder with melancholic features was identified (CFS-MDDm) group. The NF group defined as a control group whereas other four groups are defined as case groups based definition of CFS. The final goals of the experiment was to identify differentially regulated genes for the group pairs: Control (NF) vs. Case (4 groups), Control (NF) vs Case (CFS ), Control (NF) vs Case (CFS-MDDm ), Control (NF) vs Case ( ISF ), Control (NF) vs Case (ISF-MDDm ),Control (NF) vs Case (ISF-MDDm+CFS-MDDm), Control (NF) vs. Case (ISF+CFS). Findings from our study will suggest future studies needed to identify the underlying ideology of chronic fatigue syndrome and dataset used for this study presented here is not intended to focus on the intrinsic of data underlying biological interpretation. 3. Quality Control Analysis and Outlier Detection The first processing step is an outlier detection and we performed several methods to examine outlying slides. Figure 1 is the result of the principal component analysis for five groups, which identifies the distinct outlying slides visually. We saw samples 11 and 1 were separated from the dense group and could be classified as outlying slides. In addition, we examined the slides with unusual expression patterns or larger variabilities through diagnostic plots [7]. These outlying slides usually tend to have large impacts on the analysis, such as identification of differentially expressed genes. Therefore, we applied graphical method to detect outlying slides of sample. With this quality measures, we are able to compare variability among slides of samples and minimize the amount of errors made in the statistical data preparation in one-channel microarray data. Figure 2 is the diagnostic plot for detecting outlying slides. The plot shows that Slides 11, 162 and 158 seem to have quite different patterns from those of other slides. The slide 11 was identified very clearly distinctive pattern from those of other slides which was also identified by the principal component analysis. In order to confirm whether they are outlying slides, we performed the leave-one-out anlysis. Fortunately, there is no significant difference for data analysis yield after and before removed outlying slides in this study, therefore we use dataset including these outlier samples for further analysis.. 4. Normalization Figure 3 is the boxplot of the original intensity data from five individual sample groups before normalization. The first plot in Figure 4 is the group level plot for these raw data showing the 25%, 50 %, and 75% percentile for each sample of five groups. The x-axis represents the slide and the y-axis represents the the quartiles of the actual intensities. This group level plot allows us to investigate the group-specific variations within data. The group-level plot clearly showed the different levels of expression data for the different sample groups. The next processing step is normalization procedure performed by Quantile, LOWESS and IQR scale normalization methods [8-10]. Quantile normalization method give each chip the equalizing the entire distribution and quicker and simpler. Another approach is LOWESS normaliation based upon M versus A plot, where M is the difference in log expression values and A is the average of the log expression values. However, rather than being applied to two color channels on the same array, it was applied to sample intensities from two arrays at a time. Because this method works in a pairwise manner, it is somewhat time consuming [10]. Figure 4 summarizes plots after applying these normalization methods. Figure 4 shows different effects of normalization methods. In the next section, we show that these normalization methods produce quite different results for identifying differentially expressed genes. 5. Results Table 1 is the summary table for comparing different normalization methods regarding the effect on the identification of differentially expressed genes. It showed that the estimated number of significantly genes for different group pairs. The simplest statistical method for detecting differential expression is the t test for identifying differentially regulated genes for between group pairs, whereas the analysis of variance (ANOVA) test was applied for experiment to identify differentially regulated genes for the five groups in Table 1. We also applied some widely used methods for testing differential expression among genes starting with the simple fold-change criteria using volcano plot. The volcano plot (not shown here) for LOWESS normalization comparing between CFS and NF yielded asymetric fold-change distributions, and more genes appeared upregulated than downregulated due to the differences of these two group level, whereas quantile normalization produces approximately symmetric distributions. We expect that approximately the same number of genes are up and down regulated. Table 1 showed that estimated number of regulated gene in each group comparison depended on normalization methods. We did not see any clear difference in this data to determine which normalization method is more appropriate for finding the regulated gene. We begin analyzing the original data (raw data) without applying normalization and then compared with other normalization methods. The effect is most pronounced in the comparison between NF and CFS-MDDm depending on the normalization method. 6. Discussions We address key issues in evaluating the effectiveness of the basic statistical processing steps of the microarray data that can affect the final outcome of the gene expression analysis but not intended to focus on the intrinsic of data underlying biological interpretation in this study. We presented here some normalization methods and showed that different normalization methods yield different results. Most of previous normalization methods have focused on two-channel or Affymetrix-type data, therefore it is not easily be applied to our one-channel microarray data. Due to these reason, it is necessary to have some modification and slight adaptation be applied to this kind data to have reasonable results. The use of quality measures for analyzing individual outcomes can help in estimating the reliability of final microarray study results. In particular, when the gene effect is not large as in our example, the study presented here showed that microarray data normalization and each processing steps has an important effect on the final outcome especially for the identification of differentially expressed genes. It is important to test different possibilities and analyze the effects of the decision with appropriate tools for individual processing steps. Therefore, the overview of these effects is essential for the biological interpretation of gene expression measurements. References 1. B.M. Bolstad, R.A Irizarry, M. Astrand, and T.P. Speed. A comparison of normaliza-tion methods for high density oligonucleotide array data based on variance and bias. Bioinformatics, 19(2):185193, 2003. 2. D. Edwards. Non-linear normalization and background correction in one-channel cDNA microarray studies. Bioinformatics, 19(7):82533, 2003. 3. M. Futschik and T. Crompton. Model selection and efficiency testing for normaliza-tion of cDNA microarray data. Genome Biol, 5(8):R60, 2004. 4. X. Cui and G. A. Churchill. Statistical tests for differential expression in cDNA microarray experiments. Genome Biol, 4(4):210, 2003. 5. G. K. Smyth and T. Speed. Normalization of cDNA microarray data. Methods, 31(4):26573, 2003. 6. Toni whistler,Elizabeth R.Unger.Intergration of gene expression,clinical and epidemiologic data to characterize chronic Fatigue Syndrome. Journal of Translational Medicine 7. Taesung Park, Sung-Gon Yi, SeungYeoun Lee, and Jae K. Lee (2005) Diagnostic plots for detecting outlying slides in a cDNA microarray experiment, BioTechniques ,March 2005 Volume 38, pp 463-471 8.Y. H. Yang, S. Dudoit, P. Luu, D. M. Lin, V. Peng, J. Ngai, and T. P. Speed. Normal-ization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res, 30(4):e15, 2002. 9. Irizarry, R. A., Hobbs, B., Collin, F., Beazer-Barclay, Y. D., Antonellis, K. J., Scherf, U., and Speed, T. P. (2003). Exploration, normalization and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249-264. 4/2003 10. Li, C., and Wong, W. H. (2001). Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proceedings of the National Academy of Sciences 98, 31-36. 1/2003 Table1. Number of Significant Gene depend on Normalization Methods Normalization methods Siginificance level a=0.05Original dataIQR Scale QuantieLowessAnova test( 5 groups)0106Control(NF) vs Case (4 groups)0011Control(NF) vs Case (CFS)0000Control(NF) vs Case (CFS-MDDm)15161Control(NF) vs Case (ISF)0000Control(NF) vs Case (ISF-MDDm)0230Control(NF) vs Case (ISF,CFS-MDDm)2400Normalization methods Significance level a=0.1Original dataIQR ScaleQuantileLowessAnova test( 5 groups)01110Control(NF) vs Case (4 groups)0021Control(NF) vs Case (CFS)0000Control(NF) vs Case (CFS-MDDm)12222026Control(NF) vs Case (ISF)0000Control(NF) vs Case (ISF-MDDm)66331Control(NF) vs Case (ISF,CFS-MDDm)311016 Figure 1. Principal component analysis for each group Figure 2. Quality control plot for detecting outlying sample slides Fig 3. Raw data boxplot for each group and group-level plot (Before Normalization)   SHAPE \* MERGEFORMAT  Fig4. 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