The characteristic direction: a geometrical approach to identify differentially expressed genes
© Clark et al.; licensee BioMed Central Ltd. 2014
Received: 21 November 2013
Accepted: 11 March 2014
Published: 21 March 2014
Identifying differentially expressed genes (DEG) is a fundamental step in studies that perform genome wide expression profiling. Typically, DEG are identified by univariate approaches such as Significance Analysis of Microarrays (SAM) or Linear Models for Microarray Data (LIMMA) for processing cDNA microarrays, and differential gene expression analysis based on the negative binomial distribution (DESeq) or Empirical analysis of Digital Gene Expression data in R (edgeR) for RNA-seq profiling.
Here we present a new geometrical multivariate approach to identify DEG called the Characteristic Direction. We demonstrate that the Characteristic Direction method is significantly more sensitive than existing methods for identifying DEG in the context of transcription factor (TF) and drug perturbation responses over a large number of microarray experiments. We also benchmarked the Characteristic Direction method using synthetic data, as well as RNA-Seq data. A large collection of microarray expression data from TF perturbations (73 experiments) and drug perturbations (130 experiments) extracted from the Gene Expression Omnibus (GEO), as well as an RNA-Seq study that profiled genome-wide gene expression and STAT3 DNA binding in two subtypes of diffuse large B-cell Lymphoma, were used for benchmarking the method using real data. ChIP-Seq data identifying DNA binding sites of the perturbed TFs, as well as known drug targets of the perturbing drugs, were used as prior knowledge silver-standard for validation. In all cases the Characteristic Direction DEG calling method outperformed other methods. We find that when drugs are applied to cells in various contexts, the proteins that interact with the drug-targets are differentially expressed and more of the corresponding genes are discovered by the Characteristic Direction method. In addition, we show that the Characteristic Direction conceptualization can be used to perform improved gene set enrichment analyses when compared with the gene-set enrichment analysis (GSEA) and the hypergeometric test.
The application of the Characteristic Direction method may shed new light on relevant biological mechanisms that would have remained undiscovered by the current state-of-the-art DEG methods. The method is freely accessible via various open source code implementations using four popular programming languages: R, Python, MATLAB and Mathematica, all available at: http://www.maayanlab.net/CD.
Genome-wide transcriptional profiling, the parallel measurement of the expression of tens of thousands of genes, is a powerful tool which, for example, aids in the development of clinical biomarkers for disease diagnosis, reveals the heterogeneity of histologically identical cancers, and sheds light on diverse biological mechanisms. After estimating the relative or absolute expression level of all transcripts, the next step is to test statistical hypotheses . Typically, these hypotheses are concerned with the difference between two biological conditions, for example, normal verses diseased tissue, or perturbed verses unperturbed cells. One of the most important aims of such tests is to identify the genes which are mostly responsible for the difference between the biological states under investigation, the so called differentially expressed genes (DEG).
There have been a number of attempts to apply multivariate analyses to identify DEG [8, 9]. For example, Lu et al.  proposed an application of Hotellings T2 test, which is a multivariate generalization of Welsh’s t-test. However, these approaches remain little-used because they are sensitive to the fact that typically microarray or RNA-Seq gene expression profiles have fewer samples than genes. A small sample size compared to the dimensionality of the measured variables brings difficulties to the analysis . A significant step towards the resolution of such problems was the realization that variance shrinkage improves statistical power [5, 12, 13]. Also, methods that directly attempt to identify differentially expressed gene-sets as opposed to individual genes have been developed [14–19]. In addition, to increase statistical power, these approaches also attempt to facilitate biological interpretation, which can be challenging when faced with a long list of DEG [15, 20].
There are currently two main principle technologies to perform whole-genome transcriptional profiling: microarrays and RNA-Seq. The later has a number of advantages such as greater dynamic range, and an ability to measure previously unknown transcripts. The RNA-Seq technology also presents some challenges such as potential non-uniform read coverage and transcript length biases, and recently there has been a flurry of publications approaching these important issues [21–24]. One of the differences between microarray and RNA-Seq data is that microarrays result in continuous measures of expression, often log-normally distributed, whereas RNA-Seq data results in positive integer read counts with discrete probability distributions. For this reason, established methods of differential expression analysis for microarray data are not immediately transferable to RNA-Seq data but this challenge can be overcome as demonstrated by Soneson et al.  who showed how to transform RNA-Seq counts to continuous values. The approach we shall take here relies on minimal assumptions about the distributions of the data and should apply to RNA-Seq, microarray or any other similar situation where the dimensionality of the data far exceeds the sample size.
We propose a new multivariate approach called the Characteristic Direction which is better able to identify DEG than univariate approaches including the methods: fold change, SAM, the Welch’s t test, LIMMA and DESeq. Our approach naturally incorporates a regularization scheme to deal with the problem of dimensionality, and also provides an intuitive geometrical picture of differential expression in terms of a single direction. We show how this geometrical picture reliably characterizes the differential expression and also leads to some natural extensions of the approach such as improved gene-set enrichment analysis. In addition, we take advantage of a neat mathematical trick to make the Characteristic Direction method fast to compute.
Apart from being able to assess the analysis methods, these results are interesting in themselves as they show that we are able to infer information about TF and drug perturbations from expression data. For example, we find that proteins that are known to interact with the TFs and the drug targets tend to be also within the DEG. Finally, we show how a natural extension of the Characteristic Direction method can be used to perform potentially improved gene-set enrichment analysis. We compared enriched terms for DEG identified in human cancer stem cells and show that the Characteristic Direction enrichment method recovers more relevant Gene Ontology (GO) terms as compared with GO terms recovered by the hypergeometric test, or GSEA.
Computing the characteristic direction and identifying differentially expressed genes
Classification approaches, for example those that predict clinical outcome from gene expression data, are inherently multivariate as they use the structure of the gene expression profiles as a whole in order to distinguish between biological conditions or classes. Our approach is to repurpose linear classification methods in order to characterize differential expression and identify DEG. We use a linear classification scheme, which defines a separating hyper-plane; the orientation of which we show can be interpreted to identify DEG. We also find that the direction normal to the separating hyper-plane provides a simple geometrical conceptualization of the differential expression, which naturally leads to extensions of the approach, such as a new formulation of gene set enrichment analysis.
The estimation, from the data, of the terms in this equation is explained in the Additional file 1. Below we will interpret the direction of the p-vector, b, as the direction in expression space that best characterizes the differential expression, and show how the components of this vector can be used to identify differentially expressed genes. However, first we note a few potential issues: the calculation involves the inverse of a very large p × p matrix which is not only expensive to compute but also the elements must be estimated from a relatively small sample-size (p >> N), which means that the matrix is singular and this leads to large variance in the results even when using the generalized inverse.
where is the estimated covariance matrix, and σ2 is the scalar covariance (see Additional file 1 for elaboration). The inclusion of a constant on the diagonal resolves the singularity problem, and the modulation of the off-diagonal terms helps to reduce noise arising from the estimation of covariance from few samples.
Then the contribution of each to this sum can be interpreted as quantifying the relative contribution of each component to the total differential expression giving the significance of the corresponding gene. The above interpretation provides a quantitative measure of the relative, but not absolute, significance of each gene to the differential expression, and as such can be used to rank the genes in order of significance. However, we also want to identify a shortlist of significant DEGs. This could be done completely within the framework we have outlined by using a L1 regularization scheme in place of that used in the shrinkage equation above; such a penalty results in automatic feature selection because many components fall to zero; the genes corresponding to the features retained would then comprise the DEGs. An alternative method to deriving a significance threshold is described below.
Generating synthetic data
We generate synthetic normalized expression data which incorporates multivariate structure. The multivariate structure of real biological expression data is not fully known, we therefore use a simple approach which incorporates some of the best established properties of such data: 1) large number of features (genes) with a relatively small number of samples; 2) significant dependencies between the expression levels of the genes, leading to dimensionality which is much smaller than the number of features. In addition to these properties we require control over the number and identity of genes which are differentially expressed between two datasets. There are a number of ways that datasets with these properties may be generated, but we chose the simplest, with the fewest free parameters. In a nutshell, we use a multi-variate normal distribution distributed throughout a random subspace of the full expression space, the dimension of which reflects the dimension of the dataset. By ensuring that this subspace spans a predefined vector of differentially expressed genes we can perturb the mean of the normal distribution, preserving the covariance matrix, to generate data with pre-defined differentially expressed genes. An explicit description of the algorithm follows:
Estimating significant DEG applied to the synthetic data
The Characteristic Direction method is represented by a vector in expression space, each component of which corresponds to a gene. We interpret this vector by taking the square of each component to be a measure of the importance of the corresponding gene in the differential expression; the larger the squared component the more significant the gene. In order to determine the appropriate threshold above which to accept genes as differentially expressed we derive a null distribution for the ranks of the squared components as follows:
Generate two random sample means by drawing from the multivariate student t distribution with N - 1 degrees of freedom and find their difference.
Calculate the null characteristic direction b null = Σ-1 Δ m
Calculate and rank the components into descending order of magnitude
- 4.Repeat steps 1–3 100 times, and take the mean, to give
To compare the real distribution the null we take the ratio: . The simplest and most conservative approach would be to accept into the set of differentially expressed genes all those genes for which the ratio: . A less conservative method to derive the threshold from the data is to consider the inflection in the curves which can be isolated with the cumulative distributions.
Performing characteristic direction enrichment analysis
Benchmarking the characteristic direction method with transcription factor perturbations followed by microarray genome-wide expression profiling
We collected 73 experiments from GEO (Additional file 1: Table S1) which contain expression data for control verses TF perturbation with at least three biological replicates in each of these classes. The TF perturbations consisted of knockdowns (32), knockouts (29), over-expressions (5), and other types of perturbation (7) such as partial mutations for example. A complete list with the details about these experiments can be found in the Additional file 1. We extracted processed expression values from the SOFT files downloaded from the GEO database. For each experiment, we compared control and perturbed classes with four different methods: the fold change, Welsh’s t test, SAM, and the geometrical approach described above which we shall refer to as the “characteristic direction” approach. Each experiment and method pair resulted in a ranked list of all genes on the particular array chip in order of their estimated significance in the differential expression.
To evaluate the ability of each method to prioritize DEG we used ChIP-Seq data reporting DNA binding sites for the each TF from one of two databases: ChEA  and ENCODE . There is little overlap between these databases and so they constitute independent validations (see Additional file 1). Using this data we derived lists of genes which are associated with each TF by the identification that the TF bind to these genes’ promoters. Then, by assuming that genes from these lists are more likely to be regulated by the perturbed TF than the complementary genes, we reasoned that the degree to which an analysis method prioritizes the ChIP-Seq derived genes is a measure of its effectiveness. In addition, in a similar way we used lists of genes/proteins which are known to physically interact with each TF. We reasoned that genes for which their protein product physically interacts with the TF are more likely to be differentially expressed after the TF perturbation. As a final comparison, we examined the priority given to the perturbed TF itself, since it is known that many TF tend to auto-regulate their expression.
Any significant deviation from this indicates that the gene sets are significant in the differential expression analysis, therefore we examine D(r) - r for significant deviations from zero in order to evaluate the various methods. A significant positive value corresponds to the genes in S j being concentrated at the smaller scaled ranks and therefore having greater significance than a uniform random distribution. The entire process is visualized in Figure 2.
Random fluctuations from zero are to be expected and we can estimate the scale for these fluctuations, φ, by a premise similar to that behind the Kolmogorov-Smirnov test (see the Additional file 1 for details). When plotting D(r) - r we also include a right-hand scale to the plots which have the values scaled by φ to give an impression of how the deviation compares to what might be expected from random fluctuations under the null hypothesis of a uniform distribution of rankings. Values >> 1 on this scale indicate significant non-uniformity in the distribution of ranks.
Benchmarking the characteristic direction method with drug perturbations followed by microarray genome-wide expression profiling
Next we collected 130 experiments from GEO (Additional file 1: Table S2) consisting of control verses FDA approved drug perturbed samples, with at least three biological replicates in each sample. The genes were ranked in the same way as in the previous subsection, using the same methods. Due to the different mechanisms of action between TFs and drugs, instead of using ChIP-Seq for validation we assessed the rankings of known drug targets, and separately, genes which are known to have their protein products directly physically interact with those drug targets using known protein-protein interactions available from the NCBI Gene database and drug targets from DrugBank . We assess the prioritization of the genes with the DEG calling methods in the same way as for the TFs otherwise. It should be noted that ChIP-Seq data informs the validation with relatively unbiased and objective data whereas the knowledge of drug targets is rather more biased and incomplete. In addition, it is not known whether targeting a drug target with a drug will alter the mRNA expression of the target. So we do not expect to see the same strength of signal in this form of validation as compared with the validation for TF perturbation followed by expression with ChIP-Seq prior data. The performance of each method seems to be in the same relation as for the TFs, with the characteristic direction giving higher priority to the genes encoding drug targets of the relevant drugs and genes which their products interact with those targets (Figure 5e-f) (Kolmogorov-Smirnov test p-values comparing all the distributions can be found in Additional file 1: Tables S7 and S8).
Comparing the characteristic direction method to DESeq
Benchmarking the characteristic direction method with synthetic data
Estimating significant DEG applied to the synthetic data
Characteristic direction enrichment analysis
In the case study presented in this section we attempt to compare the various biological contexts that emerge when examining differentially expressed genes identified from mRNA profiling of CD44+ CD24-/low breast cancer cells as compared with normal breast epithelium tissue. The data used in this case study for evaluation and validation comes primarily from a study that profiled and compared normal breast epithelium tissue obtained from reduction mammoplasties and highly tumorigenic breast cancer cells isolated from tumors (ESA+ CD44+ CD24-/low Lin-) . The various approaches to identify DEGs from this dataset may provide different pictures of the biological mechanisms which are relevant to the disease. When comparing CD44+ CD24-/low breast cancer stem cells with normal breast epithelium tissue we expect to detect biological processes such as cell motility, cell proliferation, wound healing , and extra cellular matrix (ECM) remodeling which are known to be up-regulated in cancer stem cells and are activated in aggressive tumors.
Discussion and conclusions
We have described a new multivariate approach to differential expression which is better able to identify DEG while also addressing the issues associated with the high dimensionality of expression data. The Characteristic Direction approach uses the orientation of the separating hyperplane from a linear classification scheme, linear discriminant analysis, to define a direction which characterizes the differential expression. This results in a simple, highly-regularized characterization which is appropriate for genome-wide expression analysis. We compared the performance of this approach to established univariate approaches, with real and synthetic data. The validation scheme in the context of TF and drug perturbations is in itself valuable for benchmarking both computational and experimental methods. Extracting a large number of control verses perturbation expression datasets from GEO and prioritizing the genes with the various methods, we were able to show that the Characteristic Direction approach prioritizes genes which are associated with the binding sites of the perturbed TF and targets of drugs respectively; and it does so to significantly greater degree than a selection of popular methods. We took advantage of the opportunity to use independent prior knowledge datasets to validate our method. It is established that binding and unbinding of transcription factors to the promoters of genes is, in general, used for gene expression regulation. However, it is also clear that binding to the promoter does not necessarily result in differential expression. This is especially true when considering different cellular contexts. In most cases of our validation scheme the ChIP and array do not come from the same cell lines. However, there is some correlation/overlap between DEG after TF knockdowns and TF putative binding based on ChIP for the same TF in most cases. We do not know the true positives but we know that more overlap is likely due to a more accurate method to identify the DEG. We name this a silver standard for validation as it is not as good as a gold standard but it is good enough to compare DEG calling methods. The fact that we were able to recover genes associated with the binding sites of the perturbed TF is interesting on its own as it reveals a relation between DNA interactions identified by ChIP-Seq experiments and mRNA levels from expression profiling. Similarly, the ability of the method to identify a clear relationship between drug targets and the differential expression of their interactors in a systematic way is also noteworthy because for many drugs we do not know the targeted pathways while differential expression signatures are readily available. For the RNA-Seq validation we used a single study which compares differential binding of a TF, to differential expression, in the context of high-throughput sequencing. We found a stronger apparent relationship between differential binding and differential expression when using the Characteristic Direction approach as compared to the DESeq method. Like all statistical methods, the Characteristic Direction method works best when there are many repeats of the same condition. In principle, the method requires at least two repeats, but at least three repeats are needed for practical applications. The microarray and RNA-seq data used for validation of the method always had at least three repeats for each condition. It is true that in most RNA-seq studies so far investigators do not have that many repeats (1 or 2), but this is likely to change as the cost of such experiments rapidly drops. To make the Characteristic Direction method accessible, we implemented it in Python, R, MATLAB and Mathematica. Readers that are interested in applying the method to their own data should refer to the open source scripts and examples available at: http://www.maayanlab.net/CD.
Availability and requirements
Implementations of the method are provided in Python, R, MATLAB, and Mathematica freely available at: http://www.maayanlab.net/CD.
This work was supported in part by grants from the NIH: R01GM098316-01A1, P50GM071558, R01DK088541-01A1, U54HG006097-02S1 and RC4DK090860-01.
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