Preferred analysis methods for Affymetrix GeneChips revealed by a wholly defined control dataset
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As more methods are developed to analyze RNA-profiling data, assessing their performance using control datasets becomes increasingly important.
We present a 'spike-in' experiment for Affymetrix GeneChips that provides a defined dataset of 3,860 RNA species, which we use to evaluate analysis options for identifying differentially expressed genes. The experimental design incorporates two novel features. First, to obtain accurate estimates of false-positive and false-negative rates, 100-200 RNAs are spiked in at each fold-change level of interest, ranging from 1.2 to 4-fold. Second, instead of using an uncharacterized background RNA sample, a set of 2,551 RNA species is used as the constant (1x) set, allowing us to know whether any given probe set is truly present or absent. Application of a large number of analysis methods to this dataset reveals clear variation in their ability to identify differentially expressed genes. False-negative and false-positive rates are minimized when the following options are chosen: subtracting nonspecific signal from the PM probe intensities; performing an intensity-dependent normalization at the probe set level; and incorporating a signal intensity-dependent standard deviation in the test statistic.
A best-route combination of analysis methods is presented that allows detection of approximately 70% of true positives before reaching a 10% false-discovery rate. We highlight areas in need of improvement, including better estimate of false-discovery rates and decreased false-negative rates.
KeywordsAdditional Data File Probe Pair Nonspecific Signal Control Dataset Nonspecific Hybridization
Since their introduction in the mid 1990s [1, 2], expression-profiling methods have become a widespread tool in numerous areas of biological and biomedical research. However, choosing a method for analyzing microarray data is a daunting task. Dozens of methods have been proposed for the analysis of both high-density oligonucleotide (for example, Affymetrix GeneChip) and spotted cDNA or long oligonucleotide arrays, with more being put forward on a regular basis . Moreover, it is clear that different methods can produce substantially different results. For example, two lists of differentially expressed genes generated from the same dataset can display as little as 60-70% overlap when analyzed using different methods ( and see Additional data file 1). Despite the large number of proposed algorithms, there are relatively few studies that assess their relative performance [5, 6, 7, 8, 9]. A significant challenge to undertaking such studies is the scarcity of control datasets that contain a sufficiently large number of known differentially expressed genes to obtain adequate statistics. The comparative studies that have been performed have used a small number of positive controls, and have included a background RNA sample in which the concentrations of the various genes are unknown, preventing an accurate assessment of false-positive rates and nonspecific hybridization.
The most useful control datasets to date for evaluating the effectiveness of analysis methods for Affymetrix arrays are cRNA spike-in datasets from Affymetrix and Gene Logic. The Affymetrix Latin square dataset  is a series of transcriptional profiles of the same biological RNA sample, into which 42 cRNAs have been spiked at various known concentrations. The dataset is designed so that, when comparing any two hybridizations in the series, all known fold changes are powers of two. The Gene Logic dataset  has a similar experimental design, but with 11 cRNAs spiked in at varying fold changes, ranging from 1.3-fold upwards.
Here we present a new control dataset for the purpose of evaluating methods for identifying differentially expressed genes (DEGs) between two sets of replicated hybridizations to Affymetrix GeneChips. This dataset has several features to facilitate the relative assessment of different analysis options. First, rather than containing a limited number of spiked-in cRNAs, the current dataset has 1309 individual cRNAs that differ by known relative concentrations between the spike-in and control samples. This large number of defined RNAs enables us to generate accurate estimates of false-negative and false-positive rates at each fold-change level. Second, the dataset includes low fold changes, beginning at only a 1.2-fold concentration difference. This is important, as small fold changes can be biologically relevant, yet are frequently overlooked in microarray datasets because of a lack of knowledge as to how reliably such small changes can be detected. Third, our dataset uses a defined background sample of 2,551 RNA species present at identical concentrations in both sets of microarrays, rather than a biological RNA sample of unknown composition. This background RNA population is sufficiently large for normalization purposes, yet also enables us to observe the distribution of truly nonspecific signal from probe sets which correspond to RNAs not present in the sample.
We have used this dataset to compare several algorithms commonly used for microarray analysis. To perform a direct comparison of the selected methods at each stage of analysis, we applied all possible combinations of options to the data. Thus, it was possible to assess whether some steps are more critical than others in maximizing the detection of true DEGs. Our results show that at several steps of analysis, large differences exist in the effectiveness of the various options that we considered. These key steps are: first, adjusting the perfect match probe signal with an estimate of nonspecific signal (the method from MAS 5.0  performs best); second, checking that the log fold changes are roughly distributed around 0 (by observing the so-called M versus A plot , the plot of log fold change (M) versus average log signal intensity (A)), and if necessary, performing a normalization at the probe-set level to center this plot around M = 0; and third, choosing the best test statistic (the regularized t-statistic from CyberT  is most accurate). Overall, we find a significant limit to the sensitivity of microarray experiments to detect small changes: in the best-case scenario we could detect approximately 95% of true DEGs with changes greater than twofold, but less than 30% with changes below 1.7-fold before exceeding a 10% false-discovery rate. We propose a 'best-route' combination of existing methods to achieve the most accurate assessment of DEGs in Affymetrix experiments.
Results and discussion
The number of clones and assigned fold change for each pool of PCR products
Number of clones
Number of assigned Affymetrix probe sets
Assigned fold change (S vs C)
Amount of RNA added to each C chip (μg)
Amount of RNA added to S chip (μg)
Assignment of Affymetrix probe sets to DGC clones
In the Affymetrix GeneChip design, the expression level of each RNA species is reported by a probe set, which in the DrosGenome1 chip  comprises 14 oligonucleotide probe pairs. Each probe pair contains two 25-mer DNA oligonucleotide probes; the perfect match (or PM) probe matches perfectly to the target RNA, and the mismatch (or MM) probe is identical to its PM partner probe except for a single homomeric mismatch at the central base-pair position, and thus serves to estimate nonspecific signal.
The DrosGenome1 chip used in this experiment is based on release version 1.0 of the Drosophila genome sequence and thus does not represent the most up-to-date annotated version of the genome. To ensure that probe-target assignments are made correctly, we assigned the 14,010 probe sets on the DrosGenome1 GeneChip to the spiked-in RNAs by BLAST of the individual PM probe sequences against the Drosophila Gene Collection release 1.0 (DGC ) clone sequences that served as the template for the cRNA samples (Materials and methods). Of the 3,860 DGC clones used in this study, 3,762 (97%) have full-length cDNA sequence available at the DGC web site, 90 have 3' and 5'-end sequence only, and eight have no available sequence. For each probe set, all clone sequences with BLAST matches to PM probe sequences in that probe set are collected, allowing at most two (out of 25 base-pair (bp)) mismatches, and only allowing matches on the correct strand. If at least three PM sequences match to a given clone, then the probe set is assigned to that clone. Matches of one probe set to more than one clone are allowed. In this manner, 3,866 probe sets are assigned to at least one DGC clone each. Among these probe sets, 1,331 have an increased concentration between the S and C chips, whereas 2,535 represent RNAs with equal concentration between the two samples. Among those probe sets which do not have any assignment using this criterion, if fewer than three PM probes within the probe set have a BLAST match to any clone, the probe set is then called 'empty' (that is, its signal should correspond to nonspecific hybridization). There are 10,131 empty probe sets; combined with the 2,535 1x probe sets, about 90% of the probe sets on the chip represent RNAs with constant expression level between the C and S samples. The rest of the probe sets are then called 'mixed', meaning that they match to more than one clone, but each with only a few PM probe matches. There are only 13 mixed probe sets. The numbers of probe sets assigned to each fold-change class are depicted in Table 1.
Assessment of absent/present call metrics
Our dataset design provides the rare knowledge of virtually all of the RNA sequences within a complex sample (excepting the small number (3%) of clones for which only partial sequence was available, and the possible rare mistakenly assigned or contaminated clone). We can therefore evaluate various absent/present call metrics on the basis of their ability to distinguish between the known present and absent RNAs. We investigate this issue at both the probe pair level and probe set level. For the probe pair level assessment, we first identify the probe pairs which we expect to show signal, and those which should not. We thus define two classes of probe pairs: first, perfect probe pairs, whose PM probe matches perfectly to a target RNA sequence, and neither PM nor MM probe matches to any other RNA in the sample with a BLAST E-value cutoff of 1 and word size of 7, and second, empty probe pairs, whose PM and MM probes do not match to any RNA sequence when using the same criteria.
When signals from the 14 probe pairs in each probe set are combined to create a composite absence/presence call, a much larger fraction of the spiked-in RNA species can be detected reliably. To obtain absent/present calls at the probe-set level, we perform the Wilcoxon signed rank test using each of the metrics listed above . The p-values from this test are used to generate the ROC curves in Figure 2d. Again, the best results are obtained when the metric compares PM with MM signals, as opposed to monitoring signal alone. The metric used in MAS 5.0 ((PM-MM)/(PM+MM)), which is equivalent to log2(PM/MM), performs best. Therefore, the MM signals are important in generating accurate presence/absence calls. In our dataset, about 85% of the true positives could be detected before having a 10% false-discovery rate. The detection of perfect probe pairs is not improved when we include additional information from replicates. The 15% of probe sets which are called absent may represent truly absent RNAs, owing to failed transcription or labeling (see Additional data file 5). However, as we do not have an independent measure of failed transcription for the individual cRNA sequences in the target sample, we cannot completely rule out the possibility that they are the result of non-responsive probes or a suboptimal absent/present metric that fails to score low-abundance cRNAs. Regardless, as non-responsive probes or missing target cRNAs should affect both the C and S chips identically, these factors should not limit the value of this dataset in making relative assessments of different analysis methods.
Generating expression summary values
An estimate of the background signal, which is the signal due to nonspecific binding of fluorescent molecules or the autofluorescence of the chip surface, was generated using two possible metrics. The MAS background  is calculated on the basis of the 2nd percentile signal in each of 16 subsections of the chip, and is thus a spatially varying metric. The Robust Multi-chip Average (RMA) algorithm  subtracts a background value which is based on modeling the PM signal intensities as a convolution of an exponential distribution of signal and a normal distribution of nonspecific signal.
Normalization at the probe level
The signal intensities are normalized between chips to allow comparisons between them. Because in our dataset, a large number of RNAs are increased in S versus C (and none are decreased), commonly used methods often result in apparent downregulation for spiked-in probe sets in the 1x change category. We thus added a set of modified normalization methods which used our knowledge of the 1x probe sets. The following different methods were applied. Constant is a global adjustment by a constant value to equalize the chip-wide mean (or median) signal intensity between chips. Constantsubset is the same global adjustment but equalizing the mean intensity for only the probe sets with fold change equal to 1. Invariantset  is a nonlinear, intensity-dependent normalization based on a subset of probes which have similar ranks (the rank-invariant set) between two chips. Invariantsetsubset is the same as invariantset but the rank-invariant set is selected as a subset of the probe sets with fold change equal to 1. Loess normalization  is a nonlinear intensity-dependent normalization which uses a local regression to make the median fold change equal to zero, at all average intensity levels. Loesssubset normalization is the same as loess but using only the probe sets with fold change equal to 1. Quantile normalization  enforces all the chips in a dataset to have the same distribution of signal intensity. Quantilesubset normalization is the same as quantile but normalizes the spiked-in and non-spiked-in probe sets separately.
We chose three ways to adjust the PM signal intensities to account for nonspecific signal. The first is to subtract the corresponding MM probe signal (subtractmm). The second is the method used in MAS 5.0, in which negative values are avoided by estimating the nonspecific signal when the MM value exceeds its corresponding PM intensity . The third is PM only (no correction). The subtractmm and MAS methods are compatible only with the MAS background correction method; that is, it does not make sense to combine these with RMA background correction.
The 14 probe intensity values were combined using one of the following robust estimators: Tukey-biweight (MAS 5.0); median polish (RMA); or the model-based Li-Wong expression index (dChip). Analyses including the subtractmm PM correction method require dealing with negative values when PM is less than MM, which occurs in about a third of the cases. Within Bioconductor, the Li-Wong estimator can handle negative values, but the other two metrics mostly output 'not applicable' (NA) for the probe set when any of the constituent probe pairs has negative PM - MM. The result for MAS and median polish is NA for about 85% of the probe sets on the chip. To study the consequence of losing so many probe sets, we modified one of these two metrics (median polish) to accept negative (PM - MM) (medianpolishna), and added this metric whenever subtractmm was used.
Normalization at the probe set level
Comparison of the observed fold changes with known fold changes
We also observed that the fold changes resulting from the chips are consistently lower than the actual fold changes. Apparently, the decrease in fold change is only partly the result of signal saturation (Figure 5b-c), and is not a byproduct of the robust estimators used to calculate expression summaries (because the low fold changes are also observed at the probe pair level; see Additional data file 3). In other experiments we have also observed that our Affymetrix fold-change levels are smaller than those obtained by quantitative reverse transcription (RT)-PCR (data not shown). One likely explanation is that we do not have an adequate estimate for nonspecific signal. For example, if we choose the MM signal as the nonspecific signal (thus calculating PM - MM, or PM - CT from MAS 5.0), we are probably overestimating the nonspecific signal, as the MM intensity value responds to increasing target RNA concentrations, and therefore contains some real signal. On the other hand, if we choose not to use a probe sequence-dependent nonspecific signal (such as in RMA), we are likely to underestimate the nonspecific signal for a large number of probes. In either case, the result is decreased fold change magnitudes. Artificially low fold-change values have been noted by others, including those investigating the Affymetrix Latin square , GeneLogic  and other  datasets, although some of the differences they report are smaller than are observed here.
Test statistics and ROC curves
Comparison of options at each of the other analysis steps
Among the background correction methods, the MAS 5.0 method generally performs better than the RMA method (Figure 7b). No clearly superior normalization method was found at the probe level (Figure 7c), even when using the subset normalization variants, although quantile normalization tended to underperform in the absence of the second normalization step.
With respect to adjusting the PM probe intensity with an estimate of nonspecific signal, Figure 7d clearly shows that either subtracting the MM signal (subtractmm), or using the MAS 5.0 correction method, is better than using uncorrected or RMA-corrected PM values (PM-only). The MAS 5.0 method performs the best because it does not create any negative values. This result is in apparent conflict with the conclusions of Irizarry et al. , who show drastically reduced noise at low signal intensity levels when the PM signal is not adjusted with MM values, and therefore better detection of spiked-in probe sets when using the fold change as the cutoff criterion. However, when Irizarry et al. use a test statistic that takes the variance into account, PM-only and MM-corrected methods (MAS) have similar sensitivity/specificity (Figure 3d,e from ). In the dataset presented here, the MAS PM-correction method yields a high variance at low signal-intensity levels, which effectively reduces the false-positive calls at this intensity range when using CyberT, thus resulting in better performance than when using PM-only. We can reconcile the Irizarry et al. result with our observations by considering a major difference between the datasets used by the two studies. Both the Affymetrix and GeneLogic Latin square datasets used in  involve a small number (10-20) of spiked-in cRNAs in a common biological RNA sample, and therefore comparisons are made between two samples that are almost exactly the same. As a result, the nonspecific component of any given probe's signal is expected to be almost identical in the two samples, and should not contribute to false-positive differential expression calls. In contrast, a large fraction of our dataset is differentially expressed; in addition, the C sample contains a high concentration of (unlabeled) poly(C) RNA. Because nonspecific hybridization depends both on a probe's affinity and on the concentrations of RNAs that can hybridize to it in a nonspecific fashion, we expect that each probe's signal can have different contributions of nonspecific hybridization between the C and S chips. Figure 2a shows that nonspecific hybridization can be a large component of a probe's signal. We hypothesize that, for our dataset, PM-only performs worse than MM-corrected methods (subtractmm or MAS) because PM-only does not try to correct for nonspecific hybridization in a probe-specific fashion. In contrast, for the Latin square datasets used in , PM-only works just as well as MM-corrected methods because the contribution of nonspecific hybridization is constant. Therefore, datasets which compare substantially different RNA samples (such as two different tissue types) should probably be processed using the MAS 5.0 method for PM correction.
Figure 7e compares the different robust estimators that were used to create expression summaries. Of these, median polish (RMA) and the Tukey Biweight methods (MAS 5.0) perform the best. Figure 7f highlights the 10 best summary method option sets, which are also depicted in Figure 3, as well as straight applications of some popular software, with or without an additional normalization step at the probe-set level. The result from the MAS 5.0 software, when adjusted with the second loess normalization step, ranks among the top 10. However, the other methods (dChip, RMA and MAS 5.0 without probe-set normalization) are not as sensitive or specific at detecting DEGs.
We were concerned that some of our analyses might be confounded by a possible correlation between low fold change and low expression summary levels, which could affect the interpretations of Figure 7 (comparing different methods) and the detection of small fold changes (see below). We therefore examined the distribution of expression levels within each spiked-in fold change group, and compared the methods with respect to their ability to detect a subset of probe sets with low expression summary levels (Additional data file 5). We found that the distribution of expression levels for the known DEGs was comparable among all the fold-change groups, and that all the conclusions reported here are similarly applicable to the low expression subset. However, the sensitivity of all methods was reduced, suggesting that they perform less well on weakly expressed than on highly expressed genes. As the number of low signal spike-ins was relatively small (265 probe sets), resulting in reduced accuracy for the ROC curves, the development of additional control datasets specifically focusing on DEG detection at low cRNA concentrations will be an important extension of this study.
Models dependent on probe sequence provide a promising route to improving the accuracy of nonspecific signal measures. Here, we applied two different models (perfect match and gcrma) to the control dataset. With respect to detecting the true DEGs, these two models perform reasonably well, although slightly less well than the MAS 5.0 PM correction method. When we consider only the low signal DEGs (Additional data file 5), gcrma outperforms perfect match, and is similar in effectiveness to the top analysis option combinations.
Estimating false discovery rates
Assessment of sensitivity and specificity
As the identities and relative concentrations of each of the RNAs in the experiment were known, we were able to assess directly the sensitivity and specificity obtained by the best-performing methods. Examination of the ROC curves in Figure 7 reveals that sensitivity begins to plateau as the false discovery rate (q) increases from 10% to 30%. Taking an upper acceptable bound for q as 10%, the maximum sensitivity obtained is about 71%. Thus, under the best-performing analysis scheme, roughly 380 (29%) of the 1,309 DEGs are not detected as being differentially expressed, with the number of false positives equaling about 105. At q = 2%, sensitivity reduces to around 60%, meaning that more than 520 DEGs are missed, albeit with fewer than 20 false positives.
It is tempting to conclude from this that we are achieving adequate sensitivity in our experiments and merely need not bother with DEGs below the twofold change level. However, we would argue that obtaining greater sensitivity should be an important goal. There is ample demonstration in the biological and medical literature that small changes in gene expression can have serious phenotypic consequences, as seen both from haploinsufficiencies and from mutations that reduce levels of gene expression through transcriptional regulation or effects on mRNA stability. Furthermore, effective fold changes seen in a microarray experiment might be considerably smaller than actual fold changes within a cell, if the sample contains additional cell populations that dilute the fold-change signal. As it is often not possible to obtain completely homogeneous samples (for example, when profiling an organ composed of several specialized cell types), this is likely to prove a very real limitation to detecting DEGs. In cases where pure cell populations can be obtained, for example by laser capture microdissection, the numbers of cells are often small and RNA needs to undergo amplification in order to have enough for hybridization. Here, non-linearities in RNA amplification might also lead to observed fold changes that fall below the twofold level. We used three microarray replicates for this study, as this is frequently the number chosen by experimentalists because of cost and limiting amounts of RNA. One possible extension of this work would be to examine how many replicates are necessary for reliable detection of DEGs at a given fold change level.
We have compared a number of popular analysis options for the purpose of identifying differentially expressed genes using an Affymetrix GeneChip control dataset. Clear differences in sensitivity and specificity were observed among the analysis method choices. By trying all possible combinations of options, we could see that choices at some steps of analysis are more critical than at others; for example, the normalization methods that we considered perform similarly, whereas the choice of the PM adjustment method can strongly influence the accuracy of the results. On the basis of our observations, we have chosen a best route for finding DEGs (Figure 3). As any single choice of analysis methods can introduce bias, we have proposed a way to combine the results from several expression summary datasets in order to obtain more accurate FDR estimates. However, these estimates remain substantially lower than actual false-discovery rates, demonstrating the need for continued development of ways to assess the false-discovery rate in experimental datasets. Our analysis further revealed the existence of a high false-negative rate (low sensitivity), especially for those DEGs with a small fold change, and thus suggests the need for improved analysis methods for Affymetrix microarrays. In order to be feasible, this study investigated only a fraction of the current options. The raw data from our hybridizations are available in Additional data files 6-7 and on our websites [27, 28], and we encourage the use of this dataset for benchmarking existing and future algorithms. Also important will be the construction of additional control datasets to explore issues not well covered by the present study, such as performance of the analysis methods for specifically detecting low-abundance RNAs and the effects of including larger numbers of replicate arrays. We hope that these experiments will help researchers to choose the most effective analysis routines among those available, as well as guide the design of new methods that maximize the information that can be obtained from expression-profiling data.
Materials and methods
cRNA and hybridization
PCR products from Drosophila Gene Collection release 1.0 cDNA clones  were generated in 96-well format, essentially as described . Each PCR product includes T7 and SP6 promoters located 5' and 3' to the coding region of the cDNA, respectively. Each PCR reaction was checked by gel electrophoresis for a band of detectable intensity and the correct approximate size. Those clones which did not yield PCR product were labeled as 'failed' and eliminated from subsequent analysis. From sequence verification of randomly selected clones, we estimate the number of mislabeled clones to be < 3%. The contents of the plates were collected into 19 pools, such that each pool contained the PCR product from one to four plates (approximately 96-384 clones). Biotinylated cRNA was generated from each pool using SP6 polymerase (detailed protocol available upon request) and the reactions were purified using RNeasy columns (Qiagen). Concentration and purity for each pool was determined both by spectrophotometry and with an Agilent Bioanalyzer. The labeled products were then divided into each of two samples - constant (C) and spike (S) - at specific relative concentrations (Table 1, Figure 1). Because the C sample contains less total RNA than the S sample, 20 μg of (unlabeled) poly(C) RNA was added to the C sample to equalize the nucleic acid concentrations. By mixing the labeled pools just before hybridization, we ensured that the fold change between C and S is uniform for all RNAs within a single pool, while still allowing the absolute concentrations of individual RNAs to vary. The two samples were then hybridized in triplicate to Affymetrix Drosophila arrays (DrosGenome1) using standard Affymetrix protocols. We chose to hybridize each replicate chip from an aliquot of a single C (or S) sample, resulting in technical replication; thus this dataset does not address the noise introduced by the labeling and mixing steps. The clones comprising each pool can be found in Additional data file 8, and the resulting Affymetrix chip intensity files (.CEL) files are available in Additional data files 6-7.
Estimate of RNA concentrations
The total amount of labeled cRNA that was added to each chip (approximately 18 μg) was comparable to a typical Affymetrix experiment (20 μg). Although we do not know the individual RNA concentrations, we estimate that these span the average RNA concentration in a biological GeneChip experiment. Our biological RNA samples typically result in about 40% of the probe sets on the DrosGenome1 chip called present, so the mean amount of individual RNA is 20 μg/(14,010 × 0.40) = 0.003 μg/RNA. In the C chips, the average concentration of individual RNAs in the different pools range from 0.0008 to 0.007 μg/RNA, so the concentrations are roughly similar to those in a typical experiment. We note, however, that there is no way to ensure that the concentration distribution is truly reflective of a real RNA distribution. This is especially true with respect to the low end of the range, as it is usually unknown how many of the absent genes on an array are truly absent versus weakly expressed and thus poorly detected by the analysis algorithms used. Therefore, our analysis possibly favors methods that perform best when applied to highly expressed genes.
All of the analysis was performed using the statistical program R , including the affy and gcrma packages from Bioconductor , and scripts adapted from the hdarray library by Baldi et al. [31, 32]. In addition, we used the dChip , MAS 5.0 , Perfect Match [20, 21] and SAM  executables made available by the respective authors. Note that the false-discovery rate calculations were slightly different depending on the t-statistic variant: for the SAM statistic, false discovery rates from the authors' Excel Add-in software was used, whereas for the CyberT and basic t-statistics, the Bioconductor false-discovery rate implementation was applied, which includes an extra step to enforce monotonicity of the ROC curve. In our experience, this extra step does not qualitatively alter the results. All scripts generated in this study are available for use [27, 28].
Calculation of the statistic that combines the results of multiple expression summary datasets
Say we have n datasets and C ij , S ij are the logged signals for a given probe set in the jth C and S chips, respectively, in dataset i. The mean signal (for this probe set) for the C chips in dataset i is:
The mean fold change over all datasets is:
The modified standard deviation for the C chips in dataset i is based on the CyberT estimate:
where const is the weight for the contribution of the average standard deviation Open image in new window for probe sets with the same average signal intensity as C ij . The modified standard deviation for the S chips in dataset i (sd.S i ) is defined analogously. The pooled variance over all 10 datasets is defined as:
The variance between the 10 datasets is defined as:
Then the combined statistic was chosen to be:
Additional data files
Additional data is available with the online version of this article. Additional data file contains a figure and explanatory legend showing the degree of overlap between two lists of differentially expressed genes. Additional data file 2 lists all analysis option combinations used to generate the expression summary datasets in this study. Additional data file 3 is a plot of observed vs actual spiked-in fold changes at the probe level. Additional data file 4 shows an example of asymmetric M (log2 fold change) vs A (average log2 signal) plot for the comparison of two biological samples. Additional data file 5 contains a comparison of the analysis methods with respect to the detection of DEGs with low signal. Additional data file 6 is a Zip archive containing plain text files (in Affymetrix CEL format), Affymetrix *.CEL files for the C chips in this dataset. Additional data file 7 is a Zip archive containing plain text files (in Affymetrix CEL format), Affymetrix *.CEL files for the S chips in this dataset. Additional data file 8 contains detailed information for the individual DGC clones used in this study.
We thank M. Ramoni and M. Morrissey for helpful comments on the manuscript, K. Kerr and A. Wohlheuter for assistance with, and N. Perrimon for resources for, the PCR, the HMS Biopolymers facility for assistance with robotics and GeneChip hybridization, and B. Estrada and L. Raj for sharing the data depicted in Additional Data Files 1 and 4, respectively. S.E.C. was supported by a PhRMA Foundation CEIGI grant, a Brigham and Women's Research Council bioinformatics grant, and NIH fellowship F32 GM67483-01A1. A.M.M. is an Associate Investigator of the Howard Hughes Medical Institute. G.M.C. is supported by a PhRMA Foundation CEIGI grant. M.S.H. is supported by NIH grant K22-HG002489.
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