Leveraging twoway probelevel block design for identifying differential gene expression with highdensity oligonucleotide arrays
 Leah Barrera^{1, 2},
 Chris Benner^{1, 2},
 YongChuan Tao^{3},
 Elizabeth Winzeler^{1, 4} and
 Yingyao Zhou^{1}Email author
DOI: 10.1186/14712105542
© Barrera et al; licensee BioMed Central Ltd. 2004
Received: 23 December 2003
Accepted: 20 April 2004
Published: 20 April 2004
Abstract
Background
To identify differentially expressed genes across experimental conditions in oligonucleotide microarray experiments, existing statistical methods commonly use a summary of probelevel expression data for each probe set and compare replicates of these values across conditions using a form of the ttest or rank sum test. Here we propose the use of a statistical method that takes advantage of the builtin redundancy architecture of highdensity oligonucleotide arrays.
Results
We employ parametric and nonparametric variants of twoway analysis of variance (ANOVA) on probelevel data to account for probelevel variation, and use the falsediscovery rate (FDR) to account for simultaneous testing on thousands of genes (multiple testing problem). Using publicly available data sets, we systematically compared the performance of parametric twoway ANOVA and the nonparametric MackSkillings test to the ttest and Wilcoxon ranksum test for detecting differentially expressed genes at varying levels of fold change, concentration, and sample size. Using receiver operating characteristic (ROC) curve comparisons, we observed that twoway methods with FDR control on sample sizes with 2–3 replicates exhibits the same high sensitivity and specificity as a ttest with FDR control on sample sizes with 6–9 replicates in detecting at least twofold change.
Conclusions
Our results suggest that the twoway ANOVA methods using probelevel data are substantially more powerful tests for detecting differential gene expression than corresponding methods for probeset level data.
Keywords
gene expression analysis differential expression highdensity oligonucleotide array ANOVA FDRBackground
The use of DNA microarrays for monitoring the expression levels of thousands of genes simultaneously has generated a stream of methodological and computational challenges. In particular, the reliable identification of differentially expressed genes across different tissues, time points or treatment conditions is the most common and central task in the majority of such experiments [1]. This task has been cast as a multiple hypothesistesting problem of the simultaneous test for each gene j of the null hypothesis of no change in expression level between two or more experimental conditions. Tackling this problem generally involves the following key steps: (1) computing a test statistic for each gene j, T_{ j }and determining the significance of each test statistic using parametric assumptions or by appropriate estimation of a null distribution, and (2) employing an appropriate multiple testing procedure to determine which hypotheses to reject while controlling an appropriate error rate [2, 3].
A slew of statistical models has been developed to overcome the limitations of the classical ttest, ranksum methods, and other oneway ANOVA methods currently applied to detecting differential gene expression [4]. Under nonnormal situations the classical parametric ttest is too conservative, and like the Wilcoxon test, with its lack of distributional assumptions, suffers from low power [5]. Nonparametric variants of the ttest include the use of permuted data sets to estimate the null distribution of tstatistics for each gene [6]. With a small number of replicates, the former method suffers from coarse resolution, resulting in too few or too many genes called differentially expressed depending on the significance threshold. A mixturemodeling approach to calculate the distribution of tstatistic type scores has been proposed to overcome that limitation [6]. This approach is similar in spirit to the significance analysis of microarrays (SAM), an increasingly popular method which also uses a tstatistic type score [6]. SAM uses permutations of repeated measurements and then pools estimated null statistics for each gene to compute an overall error rate defined as the false discovery rate (FDR) for genes identified as differentially expressed [2, 7].
The false discovery rate (FDR) is the rate at which features called significant are truly null. Here, it is the expected proportion of genes erroneously identified as differentially expressed. The control of the FDR as a multiple testing procedure was proposed by Benjamini and Hochberg as a more powerful alternative to controlling the familywise error rate (FWER) when considering multiple null hypotheses simultaneously [8]. Control of the FDR implies control of the FWER when all the null hypotheses are true [9]. Bonferroni type procedures which control FWER are considered too stringent because they control the probability of making any Type I error among the hypotheses under consideration, thus rejecting too few hypotheses when identifying differentially expressed genes [3]. On the other hand, control of the FDR has been increasingly favored for highthroughput screenings such as microarray experiments, striking a balance between FWER control and the percomparisonerrorrate (PCER) control which often yields too many false positives.
The persisting high cost of microarrays, in particular of commercial highdensity oligonucleotide arrays (HDAs) such as the Affymetrix GeneChip, and the scarcity of samples in many experiments, continue to severely limit the number of replicates used per condition, and thus restrict the potential gain in statistical power of the statistical methods described above with increasing sample size. In addition, the statistical methods described above are generally applied to experiments using both cDNA microarrays and HDAs. The differences in design between the two microarray platforms have warranted different algorithms for aspects of array analysis such as gene expression level calculation, image analysis, and normalization [10].
In this light, instead of developing a new statistical method that can be generally applied to experiments using both cDNA microarrays and HDAs as those described above, we can leverage the unique design of HDAs for better differential gene expression identification. On HDAs supplied by Affymetrix, 11–20 25base oligonucleotide probes that are exact complements to different fragments of the same gene target form a probe set. Unlike cDNA microarrays, where a single intensity ratio is collected for each gene, 11–20 probelevel measurements per probe set are collected simultaneously for any single array hybridization. However, these redundant measurements are typically summarized as one value in the form of an average difference (AD) or modelbased expression index (MBEI) for the purpose of statistical analysis [11]. Using probelevel measurements in identifying differentially expressed genes and blocking on the probe in an analysis of variance (ANOVA), combined with FDR adjustment for the multiple testing problem, are the key differences between our proposed approach and previously described related methods.
Although carrying out statistics at the probelevel immediately increases the sample size by at least an order of magnitude, it is warranted due to the large and systematic differences that are known to exist among probes that survey the same gene [11]. Due to these probespecific biases, variation induced by probes is larger than that induced by array replicates [12]. The use of the probe as a blocking factor in testing for differential gene expression in a twoway ANOVA on probelevel data is thus expected to be more sensitive than previously described methods.
Chu et al. also took an ANOVA approach at the probe level, however the experimental design of their study was different from ours, which led to a more complicated model than what we propose here [13]. Chu et al. compared their method to SAM on the same data set, but identified a very different set of differentially expressed genes (Table 3 in [13]). As pointed out by other researchers, this method cannot be easily benchmarked only based on data sets of unknown positives and negatives [14]. Lemon et al. recently proposed a probelevel Logitt method that was shown to be superior to other popular probe set methods [14]. Independent from these two studies, we reached the same conclusion that using probelevel data could significantly improve the quality of resultant gene list. In addition, we demonstrate the use of a rankbased MackSkillings test, which does not depend on any distribution models required by the two parametric studies mentioned above. Furthermore, by using an FDRbased criterion, our method not only ranks genes but also suggests statistically rigorous thresholds for gene selection.
In this study, we compared both the sensitivity and specificity of parametric twoway ANOVA and the nonparametric MackSkillings test on probelevel data against the commonlyused ttest and Wilcoxon test on probeset level data. For all tests, we employed FDRcontrolling procedures described above to account for the multiple testing problem. Two public data sets are used for benchmarking purposes: the Lemon set, where thousands of genes are expected to be differentially expressed and the Affymetrix Latinsquare data set where only 14 spiked genes out of over 9000 genes on the array are expected to show real change [15–17]. We systematically tested the effects of key factors such as expression level (concentration) of the RNA transcripts, number of replicates, amount of change to be detected, in addition to the statistical methods. In almost all cases, the proposed probelevel methods outperformed previous methods based on probeset level calculations. We also found that the twoway methods are most sensitive to transcript concentration between 4 pM and 128 pM and fold change greater than two. By comparing receiver operating characteristic (ROC) curves, we demonstrated that by taking advantage of the HDA design, the twoway methods applied on only 2–3 replicates can exhibit the same high sensitivity and specificity as a SAMlike ttest with FDRcontrol using 6–9 replicates for detecting at least twofold change. Therefore, by taking advantage of the HDA design, the present limitations of oneway ANOVAtype methods can be overcome. Matlab scripts for our methods are available on http://carrier.gnf.org/publications/ProbeStatistics.
Results
We compared the performance of the commonly used oneway ANOVA methods described above, against the twoway ANOVA methods using two publicly available microarray data sets. The first set of microarray experiments involves groups of human fibroblast cells in three conditions – serum starved, serum stimulated, and a 50:50 mixture of starved/stimulated – with six replicate Affymetrix HuGeneFL arrays in each group [16]. For this set, a total of 7011 probe sets were examined per array after the preprocessing steps. The second data set is the Affymetrix Latin Square Data for Expression Algorithm Assessment [17]. In 11 experiments (denote these as experiments AK), 14 groups of human gene transcripts in 14 different known concentrations were spiked into a background RNA mixture and hybridized to 3 replicate microarrays. In two additional experiments (denote these as experiments L and M), the same Latin Square design is followed but 12 instead of 3 replicates were used per condition. In the following study, we tracked only 12 of 14 genes due to errors in the original data set for two of the probe sets. Transcript concentrations for each spiked gene ranged from 0 to 1024 pM over the various experiments [15]. For this data, the Affymetrix HG_U95A array is used and a total of 9024 probe sets in each array were analyzed as described in the following sections after the preprocessing step.
Sensitivity
We first assessed the relative sensitivity of the statistical tests by comparing the number of genes identified as differentially expressed when controlling the FDR using either an LSU procedure or a resamplingbased approach. We compared the serum starved and serum stimulated data sets between which, the expression levels of a large number of genes were expected to vary significantly [16]. We randomly sampled three replicates per condition to make the results comparable to later analyses for which only three replicates are available. The process was repeated 100 times and results were averaged.
To assess whether we are detecting biologically meaningful change, we also applied the statistical tests on random pairs of combinations of data values within the same treatment condition, i.e. comparing serum starved samples or serum stimulated samples among themselves, respectively. The dashed lines in Fig. 1 and Fig. 2 show that as expected all methods do not identify any genes as differentially expressed within a reasonable level of FDR control. The result ensures that the extra sensitivity of the proposed twoway methods was not gained at the expense of sacrificing robustness. The specificity of the methods will be further studied later.
To study the necessity of explicitly modeling the probetreatment interaction in our ANOVA model, Ftests were applied to all genes. 5151 out of 7009 genes (73%) tested had Pvalues less than 0.05, even after a Bonferroni adjustment. A possible explanation is that the interaction term captures the changes in probe crosshybridization properties caused by the large differences in the mRNA content between the two sample groups.
Percentage of identical genes called significant by pairs of procedures
MackSkillings; LSU  ttest; LSU  Wilcoxon; LSU  

twoway;LSU  96  93   
MackSkillings; LSU  88    
ttest; LSU   
A representative run of the probelevel Logitt method [14] on the Lemon data set identified 1032 genes as differentially expressed when controlling the LSUFDR at level q = 0.05 as above – less than half the number identified by the twoway methods. The Logitt method demonstrated a level of sensitivity similar to ttest (Fig. 8) [see Additional File 1].
Effect of concentration and fold change
With as little as three replicates, we see in Fig. 3 that the parametric twoway ANOVA and the MackSkillings test are very sensitive to twofold changes when testing within a maximum concentration range of 4 to 128 pM (Fig. 3a). With a fourfold change, the twoway methods are able to successfully detect nearly all spiked gene transcripts in all pairs of experiments at FDR level q = 0.05 with the exception of one changing from 0.25 to 1 pM (Fig. 3b). Only with an eightfold change and maximum concentration between 32 and 128 pM do we begin to detect the spiked genes when using the ttest and controlling the FDR at q = 0.05 (Fig. 3c). These differences may explain the higher sensitivity of the two way methods shown in Figs. 1 and 2.
Specificity and Sample Size Effect
Discussion
In the previous sections, we compared the application of twoway ANOVA methods on probelevel data to standard statistical methods on probeset level data in identifying differential gene expression in microarray experiments. We aimed to show the importance of leveraging HDA design in the choice of statistical test and not discarding information by working with a probeset summary or average of probelevel data.
Using twoway ANOVA methods, we systematically accounted for probespecific biases in hybridization or measurement efficiency, and thus achieved higher sensitivity and specificity compared with the ttest in the range of conditions investigated with varying levels of sample size, fold change, and maximum spikein concentration. In the Lemon serumstarved and serumstimulated data set, the twoway methods coupled with LSUFDR control identified more than twice as many genes as differentially expressed compared with the ttest. With the Latin Square data set, we confirmed the specificity of the twoway methods by analyzing the ROC curves and observed that with as few as three replicates, the twoway ANOVA has a 91% sensitivity with a 99.84% specificity.
Parametric methods are commonly criticized for their lack of sensitivity and specificity when detecting differential gene expression. However, we discovered that the use of an LSU FDRcontrolling procedure with the parametric twoway ANOVA method yielded the most promising results in terms of higher sensitivity and specificity for detecting differentially expressed genes. The outstanding performance of the parametric twoway ANOVA with the LSU FDRcontrolling procedures relative to the other combinations of nonparametric tests and the resamplingbased FDR in our study suggests that in the case of gene expression analysis with HDAs, there is a substantial gain in power by working with probelevel data, and that proper treatment of this data by appropriate normalization procedures and the application of appropriate transformations (logarithm, square root) can allow us to maintain assumptions critical to the method chosen.
Even without parametric assumptions, the advantage of treating the probe as a blocking factor was clearly demonstrated by the results using the MackSkillings test. Thus, if more conservative estimates from a twoway ANOVA analysis are desired, we can choose to use the results from the nonparametric MackSkillings test and still have a substantial gain in power over the ttest. The same Affymetrix Latin Square data set has been recently studied by Lemon et al. using a probelevel Logitt method and a low false positive rate of 0.03% was achieved at the sensitivity of 87% [14]. The parametric twoway ANOVA achieved essentially the same performance and the nonparametric MackSkillings method showed an even better false positive rate of 0.01% with the same sensitivity http://carrier.gnf.org/publications/ProbeStatistics.
The power of a statistical test is a function of its sensitivity and this further depends on (1) the magnitude of the real difference to be measured, (2) the noise level or standard deviation of sample measurements, (3) the significance level at which the tests are done, and (4) the sample sizes [19]. Limitations inherent to the technology platform and suboptimal data preprocessing procedures can reduce the magnitudes of the real differences being measured. As we observed, there is a nonlinear relationship between expression values and the actual spikein concentrations at the lower and higher ends of the concentration spectrum for the Latin Square data set due to detection and measurement saturation issues [20]. With the use of probelevel data in a twoway ANOVA, we take advantage of informative probelevel differences between treatments and eliminate noise due to probe efficiency differences. In this way, the twoway ANOVA methods are better able to discern treatment differences. Control of the third factor depends on the number of false leads that one is willing to incur and this in turn varies with the goals of the experiments. Finally, the control of the fourth factor is limited by resource constraints, and in microarray experiments, this continues to be a key issue due to the costs of microarrays (two to three per condition in most labs) and availability of samples to be analyzed.
It is wellknown that increasing sample size increases sensitivity for all statistical tests, and given enough samples, one can discern biologically meaningful changes well below the differences currently measured. That we can detect differentially expressed genes using twoway ANOVA methods with only two or three replicates and get comparable results with the use of the ttest on at least 6–9 replicates is evidence of the higher power of these methods on probelevel data all other factors being equal.
In this study, we have shown that coupled with an easily implemented linear stepup (LSU) FDRcontrolling procedure, parametric and nonparametric twoway ANOVA methods using probelevel data are substantially more powerful tests than standard methods applied to probeset level data for detecting differential gene expression. Their advantage in power is especially pronounced when working with samples with as few as two or three replicates – the most common sample sizes for microarray experiments [1]. Although we only examined two sets of conditions in our data sets, the twoway ANOVA is a general design which easily handles other array experiment setups with two or more levels of treatments or time series points. As a wellknown and extensively used statistical method in many fields, the twoway ANOVA has inspired a body of literature for dealing with many special cases, such as unequal group sizes due to missing data from replicates, which frequently occur in microarray experiments [21, 22]. Clearly, the ease of implementation of the twoway ANOVAtype methods coupled with LSUFDR control, and the results shown herein, strongly suggest its use and further development for identifying differentially expressed genes.
Methods
For the following study, we use methods focusing on the twosample case. We briefly describe the four wellknown statistical tests and the two forms of FDR control employed in our study. Since the parametric statistical tests require the key assumption of equal group variances, logarithms of probelevel intensities and summarized expression values were taken to provide a better approximation [23].
Statistical tests
Parametric ttest
The tstatistic and its variants are powerful measures for detecting differential expression because they permit selection of genes with maximal difference in mean level of expression between two groups and minimal variation of expression within each group [4]. Here we employ the classical ttest which is a statistically equivalent test to the parametric oneway ANOVA in the twosample case [22]. As done in Reiner et al., we obtain the pvalues directly using the tdistribution with appropriate degrees of freedom depending on the sample sizes [9].
Wilcoxon rank sum test
For each gene, the distributionfree rank sum test transforms the sorted gene expression values across experiments into ranks and then tests the null hypothesis of equality of the means of the ranked values between experimental conditions [6]. For small sample sizes, exact pvalues can be obtained from precalculated statistical tables. A normal approximation of standardized test statistics is typically used to obtain pvalues for larger sample sizes. In this case, it was used for samples of size 9 and greater.
Twoway ANOVA
The use of ANOVA in testing for the equality of group means relies on the computation of the ratio of the mean square variation among group means to the mean square variation within groups. A large ratio indicates a significant difference between group means. The oneway ANOVA model, a generalization of the ttest reliably detects differences between group means only when other factors, which can cause large variation within groups, are controlled.
In the case of HDAs, probelevel intensities are a source of large and systematic variation. Thus, instead of using the summarized expression indices for each probe set for hypothesis testing and ignoring individual probe effects, we use intensity values for each probe in a probe set and control for probespecific biases by considering probe type as a blocking factor in a twoway ANOVA. For each probe set, replicate measurements of logtransformed probelevel intensities for each probe are segregated into blocks across the treatment conditions. Two types of hypothesis tests can be performed in this case: (1) the test of the equality of probe or block means to assess the significance of explicitly modeling probelevel effects, and (2) the test of equality of treatment means having accounted for variation caused by individual probes. The ANOVA model is:
Y_{ ijk }= μ + P_{ i }+ T_{ j }+ PT_{ ij }+ ε_{k(ij)},
where Y_{ ijk }is the logarithm of the probelevel intensity measurement, μ is the overall mean, P_{ i }is the effect of the probe i, T_{ j }is the effect of treatment j, PT_{ ij }is the effect of the interaction between the probe i and treatment j, and ε_{k(ij)}is the error. The probetreatment interaction term is necessary based on our results on the Lemon data set (see Results for details).
In the first test we can measure the ratio of the mean square variation among blocks to the mean square variation within groups, where each group is a treatment/block combination. The significance of these probelevel differences have been documented and were again confirmed by the extremely low pvalues associated with block effects in our study [11]. However, it is not of particular interest that measured intensities for probe A differ significantly from those of probe B in a probe set when testing for differential gene expression [12]. Here we only measure the amount of such fluctuations and remove it from the estimate of within group variability. In the second test, the test of interest for identification of differential gene expression, we measure the ratio of the mean square variation among treatments to the mean square variation within treatment/block groups. The pvalues corresponding to the ratios for the second test are determined using an Fdistribution whose numerator has degrees of freedom equal to k1 where k is the number of treatments, and whose denominator has pk(r1) degrees of freedom, where p is the number of probes in the probe set and r is the number of replicates. In this study, we maintain the assumption of equal group sizes because there are corresponding probes for each probe set across experimental samples profiled using the same array type, and in the data sets used, there are equal numbers of replicates [22, 23]
MackSkillings test
This distributionfree alternative to the classic twoway ANOVA model above transforms the probelevel intensities into ranks for each probe across the samples (replicates and conditions). It is a generalization of the nonparametric Friedman test when there are replicates. This test of no change across experimental conditions uses the MackSkillings statistic to measure the squared deviation of the sum of the ranks across the probes in a probe set for each treatment condition, from the expected sum based on no treatment differences. As with the Wilcoxon test, the exact pvalues for small sample sizes can be found in statistical tables or computed numerically. Largesample approximation allows the estimation of pvalues using a chisquare distribution with k1 degrees of freedom, where k is the number of experimental conditions [21].
FDR control
Linear stepup (LSU) procedure
The linearstep up (LSU) procedure originally described by Benjamini and Hochberg (1995) controls the FDR rate at level q by rejecting all hypotheses H_{(i)}, i = 1,...,k where are the ordered pvalues. Here, we compute the multiplicity adjusted pvalues:
and thus associate an FDR for each hypothesis test [9].
Resamplingbased procedure
Resamplingbased methods seek to gain more power by utilizing the empirical dependency structure of the data to construct more powerful FDRcontrolling procedures [3, 9]. Here we generate an m × n matrix of resamplebased pvalues [p_{ ik }] for m probe sets using n permutations of treatment labels (n = 100 in this study). We naively estimate a resamplingbased FDR for each probe set by ordering the observed pvalues P_{ j }and starting with the largest pvalue P_{(m)}we compute:
V is the average number of assumed null pvalues from all permutations as extreme as the observed value under consideration, whereas R is the number of observed pvalues as extreme as the same value under consideration. The ratio of these values gives an estimate of the FDR associated with the rejection of the hypothesis under consideration.
The statistical tests described above were performed using Matlab. Builtin Matlab functions were used to compute the test statistics and associated pvalues, and FDR adjustments were implemented as described above.
Data preprocessing
Microarray intensity normalization and gene expression calculations were performed using dChip [11]. Probe values were first normalized and their background intensities subtracted. Probe set expression values were computed using the PMonly model for expression using standard outlier detection. An additional normalization step was used to adjust the probe set expression values of each array to a median expression level of 200. Aside from the previously published advantages for using only PM probes intensity calculations using only PM probes tend to result in higher values and few if any negative values, alleviating complications when log transforming the data [11, 10]. In addition to the preprocessing using dChip, we also filtered the probe sets so that at least one sample group has an average expression level of 20. This is done in order to prevent comparing expression levels of genes that are either insignificantly expressed in both treatment conditions or are expressed at the noise level.
Additional Files
Additional file 1
File name Figure 8
File type PDF
Description of the file: Number of probe sets identified by Logitt method in the Lemon data set.
Number of probe sets called significant versus LSUadjusted FDR in the Lemon data set computed with ttest, Wilcoxon test, Logitt method, parametric twoway ANOVA and nonparametric MackSkillings method. Dashed lines indicate the control versus control comparisons.
Abbreviations
 ANOVA:

Analysis of variance
 FDR:

false discovery rate
 HDA:

highdensity oligonucleotide array
 ROC:

receiver operating characteristic
 LSU:

linear stepup procedure
Declarations
Authors’ Affiliations
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