Dataset Information

EBSeq: an empirical Bayes hierarchical model for inference in RNA-seq experiments.

ABSTRACT: Messenger RNA expression is important in normal development and differentiation, as well as in manifestation of disease. RNA-seq experiments allow for the identification of differentially expressed (DE) genes and their corresponding isoforms on a genome-wide scale. However, statistical methods are required to ensure that accurate identifications are made. A number of methods exist for identifying DE genes, but far fewer are available for identifying DE isoforms. When isoform DE is of interest, investigators often apply gene-level (count-based) methods directly to estimates of isoform counts. Doing so is not recommended. In short, estimating isoform expression is relatively straightforward for some groups of isoforms, but more challenging for others. This results in estimation uncertainty that varies across isoform groups. Count-based methods were not designed to accommodate this varying uncertainty, and consequently, application of them for isoform inference results in reduced power for some classes of isoforms and increased false discoveries for others.Taking advantage of the merits of empirical Bayesian methods, we have developed EBSeq for identifying DE isoforms in an RNA-seq experiment comparing two or more biological conditions. Results demonstrate substantially improved power and performance of EBSeq for identifying DE isoforms. EBSeq also proves to be a robust approach for identifying DE genes.An R package containing examples and sample datasets is available at http://www.biostat.wisc.edu/kendzior/EBSEQ/.Supplementary data are available at Bioinformatics online.

SUBMITTER: Leng N

PROVIDER: S-EPMC3624807 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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EBSeq: an empirical Bayes hierarchical model for inference in RNA-seq experiments.

Leng Ning N Dawson John A JA Thomson James A JA Ruotti Victor V Rissman Anna I AI Smits Bart M G BM Haag Jill D JD Gould Michael N MN Stewart Ron M RM Kendziorski Christina C

Bioinformatics (Oxford, England) 20130221 8

<h4>Motivation</h4>Messenger RNA expression is important in normal development and differentiation, as well as in manifestation of disease. RNA-seq experiments allow for the identification of differentially expressed (DE) genes and their corresponding isoforms on a genome-wide scale. However, statistical methods are required to ensure that accurate identifications are made. A number of methods exist for identifying DE genes, but far fewer are available for identifying DE isoforms. When isoform D ...[more]

PMID: 23428641

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Project description:BackgroundMicroarray data enables the high-throughput survey of mRNA expression profiles at the genomic level; however, he data presents a challenging statistical problem because of the large number of transcripts with small sample sizes that are obtained. To reduce the dimensionality, various Bayesian or empirical Bayes hierarchical models have been developed. However, because of the complexity of the microarray data, no model can explain the data fully. It is generally difficult to scrutinize the irregular patterns of expression that are not expected by the usual statistical gene by gene models.ResultsAs an extension of empirical Bayes (EB) procedures, we have developed the β-empirical Bayes (β-EB) approach based on a β-likelihood measure which can be regarded as an 'evidence-based' weighted (quasi-) likelihood inference. The weight of a transcript t is described as a power function of its likelihood, fβ(yt|θ). Genes with low likelihoods have unexpected expression patterns and low weights. By assigning low weights to outliers, the inference becomes robust. The value of β, which controls the balance between the robustness and efficiency, is selected by maximizing the predictive β₀-likelihood by cross-validation. The proposed β-EB approach identified six significant (p<10⁻⁵) contaminated transcripts as differentially expressed (DE) in normal/tumor tissues from the head and neck of cancer patients. These six genes were all confirmed to be related to cancer; they were not identified as DE genes by the classical EB approach. When applied to the eQTL analysis of Arabidopsis thaliana, the proposed β-EB approach identified some potential master regulators that were missed by the EB approach.ConclusionsThe simulation data and real gene expression data showed that the proposed β-EB method was robust against outliers. The distribution of the weights was used to scrutinize the irregular patterns of expression and diagnose the model statistically. When β-weights outside the range of the predicted distribution were observed, a detailed inspection of the data was carried out. The β-weights described here can be applied to other likelihood-based statistical models for diagnosis, and may serve as a useful tool for transcriptome and proteome studies.

Project description:BackgroundThe small sample sizes often used for microarray experiments result in poor estimates of variance if each gene is considered independently. Yet accurately estimating variability of gene expression measurements in microarray experiments is essential for correctly identifying differentially expressed genes. Several recently developed methods for testing differential expression of genes utilize hierarchical Bayesian models to "pool" information from multiple genes. We have developed a statistical testing procedure that further improves upon current methods by incorporating the well-documented relationship between the absolute gene expression level and the variance of gene expression measurements into the general empirical Bayes framework.ResultsWe present a novel Bayesian moderated-T, which we show to perform favorably in simulations, with two real, dual-channel microarray experiments and in two controlled single-channel experiments. In simulations, the new method achieved greater power while correctly estimating the true proportion of false positives, and in the analysis of two publicly-available "spike-in" experiments, the new method performed favorably compared to all tested alternatives. We also applied our method to two experimental datasets and discuss the additional biological insights as revealed by our method in contrast to the others. The R-source code for implementing our algorithm is freely available at http://eh3.uc.edu/ibmt.ConclusionWe use a Bayesian hierarchical normal model to define a novel Intensity-Based Moderated T-statistic (IBMT). The method is completely data-dependent using empirical Bayes philosophy to estimate hyperparameters, and thus does not require specification of any free parameters. IBMT has the strength of balancing two important factors in the analysis of microarray data: the degree of independence of variances relative to the degree of identity (i.e. t-tests vs. equal variance assumption), and the relationship between variance and signal intensity. When this variance-intensity relationship is weak or does not exist, IBMT reduces to a previously described moderated t-statistic. Furthermore, our method may be directly applied to any array platform and experimental design. Together, these properties show IBMT to be a valuable option in the analysis of virtually any microarray experiment.

Dataset Information

EBSeq: an empirical Bayes hierarchical model for inference in RNA-seq experiments.

Publications

EBSeq: an empirical Bayes hierarchical model for inference in RNA-seq experiments.

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