 Methodology Article
 Open Access
 Published:
Power analysis for RNASeq differential expression studies using generalized linear mixed effects models
BMC Bioinformatics volume 21, Article number: 198 (2020)
Abstract
Background
Power analysis becomes an inevitable step in experimental design of current biomedical research. Complex designs allowing diverse correlation structures are commonly used in RNASeq experiments. However, the field currently lacks statistical methods to calculate sample size and estimate power for RNASeq differential expression studies using such designs. To fill the gap, simulation based methods have a great advantage by providing numerical solutions, since theoretical distributions of test statistics are typically unavailable for such designs.
Results
In this paper, we propose a novel simulation based procedure for power estimation of differential expression with the employment of generalized linear mixed effects models for correlated expression data. We also propose a new procedure for power estimation of differential expression with the use of a bivariate negative binomial distribution for paired designs. We compare the performance of both the likelihood ratio test and Wald test under a variety of simulation scenarios with the proposed procedures. The simulated distribution was used to estimate the null distribution of test statistics in order to achieve the desired false positive control and was compared to the asymptotic Chisquare distribution. In addition, we applied the procedure for paired designs to the TCGA breast cancer data set.
Conclusions
In summary, we provide a framework for power estimation of RNASeq differential expression under complex experimental designs. Simulation results demonstrate that both the proposed procedures properly control the false positive rate at the nominal level.
Background
RNASeq has become a popular tool for studying dynamics of gene function through transcriptomic data of individuals with multiple samples of different origins [1, 2], diverse cell types [3, 4], and multiple time points [5, 6] over the past decade. The transcriptomic measurements from multiple samples of the same individual are correlated in nature. It is very challenging to estimate power in designing these RNASeq experiments as well as to do comparative analysis. Currently there is a need of developing statistical methods for sample size calculation and power estimation with correlated RNASeq data.
Since the emergence of RNASeq data, several papers have used Poisson or negative binomial (NB) distribution to model countbased expression data [7–9]. But these methods are based on generalized linear models with fixed effects, so they can not be directly applied to correlated expression data. To overcome this limitation, others proposed the generalized linear mixed effects model (GLMM) to model countbased expression data by adding random effects to allow diverse correlation structures while still assuming a Poisson distribution (PoissonLMM) or NB distribution (NBLMM) [10–12]. In addition, the bivariate negative binomial (BNB) distribution was introduced to model paired counts of brain lesions [13]. The BNB distribution is a compound distribution of two conditionally independent Poisson random variables for modeling paired counts with a Gamma random variable for modeling individual effects. Even though the BNB distribution has not yet been used for RNASeq data analysis, it is a great candidate for experiments using paired designs.
Several studies provide methods for sample size calculation and power estimation at the marginal level [14–17] or data set level [18–20] for testing differential expression of RNASeq experiments. However these methods were designed for experiments using independent samples and can not be directly applied to correlated expression data since they may lead to biased estimators of model parameters and result in a failure of proper error rate control. So far, there is lack of general methods for power analysis that can be applied to correlated RNASeq data. To overcome this deficiency, we are the first group to propose a BNB approach for paired designs and a more general GLMM approach for designs with diverse correlation structures. To ensure the false positive rate is properly controlled at the nominal level, we employ our previously published procedure for simulating the null distribution of test statistics [17] and also compare it against the asymptotic Chisquare distribution. To demonstrate performance of the new BNB and GLMM approaches, simulations were conducted under a variety of scenarios. A real TCGA data set was used for method application.
Methods
BNB model
The BNB distribution can be obtained by compounding two conditionally independent Poisson random variables XG=g∼Poisson(μg) and YG=g∼Poisson(γμg) with a Gamma random variable G∼Gamma(ϕ^{−1},ϕ). The probability mass function for the joint distribution of (X,Y) is
Without loss of generality, we use γ to denote the fold ratio of a gene between two conditions. We are interested in testing hypothesis H_{0}:γ=γ_{0} vs. hypothesis H_{1}:γ≠γ_{0}. A Wald test for testing log transformed γ is H_{0}:log(γ)=log(γ_{0}) vs. H_{1}:log(γ)≠log(γ_{0}). The likelihood ratio test (LRT) statistic and the Wald test statistic for the above hypothesis with BNB distribution are defined in Rettiganti and Nagaraja [13].
GLMM
Poisson or NB distribution is modeled through the log link function of a linear predictor of mixed effects as
where β are fixed effects and b are random effects following normal distributions. For inference on fixedeffects β, hypotheses H_{0}:Lβ=0 vs.H_{1}:Lβ≠0 is tested by LRT or Wald test. Random effects b can be tested by zstatistic for difference from 0.
Empirical parametric test
In this study, we used our previously published simulationbased empirical parametric test for inferences [17] and the extended Bonferroni method for controlling per comparison error rate (PCER) [21].
Procedure for power estimation

1.
Specify all input parameters: sample size per condition n; mean expression μ; dispersion ϕ; fold ratio γ between conditions, nominal false positive rate α, number of simulations T.

2.
Simulate count data T times from BNB(μ,γ,ϕ) under both the null and alternative hypotheses using the input parameters listed in Step 1.

3.
Fit BNB model or GLMM and obtain test statistics (LRT or Wald) under the null hypothesis for each simulation run.

4.
Calculate the 100(1−α)th percentile of the empirical null distribution of test statistics (LRT or Wald) as the critical value.

5.
Fit BNB model or GLMM and obtain test statistics (LRT or Wald) under the alternative hypothesis for each simulation run.

6.
Calculate power (percent of rejections under the alternative hypothesis) for the input parameters listed in Step 1.
Results
Simulations
Parameter setting
Count data were simulated from a PoissonGamma (BNB) distribution under two experimental conditions (e.g. baseline vs. treatment) for n subjects. The input parameters for power calculation at the marginal level are sample size n, mean expression μ at baseline, fold ratio γ between the two conditions, dispersion ϕ, and nominal false positive rate α. Parameter values for each are \(n=5, 10, 15, 20, 25; \mu =3, 5, 10, 20, 100; \gamma =\frac {1}{3},\frac {1}{2},1,2,3; \phi =0.01, 0.1, 1, 10, 100; \alpha =0.01, 0.005, 0.001, 0.0005\). Under PoissonLMM and NBLMM models, experimental condition is the fixed effect and subject is the random effect. Under each scenario, 20,000 simulations were run as described in section ‘Procedure for Power Estimation’.
Power analysis
Figure 1 shows QQ plots for both LRT and Wald test statistics under the BNB, PoissonLMM, and NBLMM models at the null hypothesis with n=5,10,15,20,25; μ=10; ϕ=1. The null distribution of LRT statistics under the BNB model can be approximated to a Chisquare distribution with 1 degree of freedom, but Wald test statistics under the BNB model are slightly above the Chisquare distribution. The null distribution of LRT statistics under the PoissonLMM model can be approximated to a Chisquare distribution with 1 degree of freedom, but LRT statistics under the NBLMM model are moderately below the Chisquare distribution. Wald test statistics under both the PoissonLMM and NBLMM models have unusually large values due to computational instability and dramatically deviate from the Chisquare distribution, therefore these two tests are not pursued further for power analysis.
Figure 2 shows the critical values of the empirical null distribution for the four tests (LRT under the BNB model, Wald under the BNB model, LRT under the PoissonLMM model, and LRT under the NBLMM model) and the critical values of the Chisquare distribution with 1 degree of freedom over 5 different sample sizes, 5 different mean expression levels, and 5 different dispersion parameters. At each input parameter setting, the false positive rate is controlled at the nominal level α=0.001 by using the empirical parametric test or the Chisquare distribution. Both the LRT under the BNB model and the LRT under the PoissonLMM model with the use of the empirical parametric test have similar critical values as the Chisquare distribution. However the Wald test under the BNB model with the use of the empirical parametric test has larger critical values than the Chisquare distribution especially at smaller mean expression levels. The LRT under the NBLMM model with the use of the empirical parametric test has lower critical values than the Chisquare distribution. When the approximated Chisquare distribution is used instead of the empirical parametric test (Fig. 3), actual false positive rates of the LRT under the BNB model and the LRT under the PoissonLMM model are well maintained at the nominal false positive rate as expected. Actual false positive rates of the Wald test under the BNB model are much larger than the nominal false positive rate especially at smaller mean expression levels. Actual false positive rates of the LRT under the NBLMM model are uniformly lower than the nominal false positive rate.
Power at two different fold ratios (2 fold down or 2 fold up respectively) for the four tests at α=0.001 are displayed in Figs. 4 and 5. In general, power increases with larger sample sizes, larger mean expression levels, and larger absolute fold changes for all four tests. The LRT under the BNB model and the LRT under the PoissonLMM model have equivalent power over different parameter values. The Wald test under the BNB model has lower power at 2 fold down and higher power at 2 fold up in general when compared to the LRT under the BNB model and the LRT under the PoissonLMM model. The LRT under the NBLMM model has lower power at almost all parameter values when compared to the LRT under the BNB model and the LRT under the PoissonLMM model.
Applications
TCGA data set
To study and demonstrate the proposed power estimation procedure in a real data application, we used the TCGA breast cancer data set as a pilot data set for designing a new study to detect differential expression using a paired design. The TCGA breast cancer data set BRCA (acquired in Feb. 2019 from FireBrowse) contains 1,212 tumor samples and 20,531 genes with nonzero counts. TMM method was used for normalization of all samples [22]. We chose the comparison between primary tumor samples and their matched normal samples (112 samples) for this case study. BNB model was fit to obtain parameter estimates for each gene. The estimated mean expression levels of matched normal samples are 14, 681, and 3,022 at the 20th, 50th, and 80th percentiles of all genes respectively. The estimated dispersion values are 0.07, 0.2, and 1 at the 20th, 50th, and 80th percentiles of all genes respectively. To demonstrate how to estimate power, we choose the mean expression level at the 20th percentile, the dispersion at the 80th percentile, and the detectable fold ratio of 2 and 0.5 between primary tumor and matched normal conditions to simulate test statistics under both the null and alternative hypotheses for 20,000 times. The LRT and Wald tests under the BNB model and the LRT under the PoissonLMM model were compared with the use of the empirical parametric test as well as the Chisquare distribution under the null hypothesis at the nominal false positive rate of 0.001. Actual false positive rates using the Chisquare distribution at this α level are shown in Fig. 6. We observe that actual false positive rates are inflated at most sample sizes for the Wald test, which is consistent with the simulation results. Figure 7 shows power as a function of sample sizes at the mean expression level of 14 and the dispersion of 1 for a 2fold up or downregulation of primary tumor compared to matched normal using the empirical parametric test for both tests. The LRT has better performance than the Wald test in general. To design a new RNASeq study with at least 80% power for the LRT, we will need n=5 samples per group to detect a 2fold upregulation or n=9 samples per group to detect a 2fold downregulation for genes at the mean expression level of 14 and the dispersion of 1. Results for all combinations of the mean expression level and the dispersion values are included in the additional file 1. The computation time for the BNB models and PoissonLMM model are about 34 and 15 hours respectively on a standard windows laptop with Intel Core i76820HQ CPU at 2.70GHz and 32GB RAM. Two main factors for the used computation time in this case are the number (i.e. 20,000) of simulations and the number (i.e. 11) of different sample sizes.
Discussion
Several studies discovered excessively inflated false positive rates for differential expression detection by using popular NB methods (i.e. edgeR, DESeq2) in RNASeq data analysis [23–26]. A reasonable explanation for this phenomena is that these methods mainly rely on biased asymptotic Chisquare distribution for inferences, which results in the failure in false positive rate control, especially in experiments with small sample sizes. In consistency with this phenomena, our published paper shows the downward bias of critical values when an asymptotic Chisquare distribution is applied for both the LRT and Wald tests under the NB model in power analysis for RNASeq differential expression studies [17]. In that paper, we provided a solution so that false positive rates can be controlled at the nominal level with the use of the empirical parametric test for obtaining critical values of both tests. In this paper, we show that either the empirical parametric test or asymptotic Chisquare distribution can be used to obtain critical values for both the LRT under the BNB model and the LRT under the PoissonLMM model. However the empirical parametric test has to be used for both the Wald test under the BNB model and the LRT under the NBLMM model because the test statistics deviate from the asymptotic Chisquare distribution.
We observed that the LRT under the BNB model and the LRT under the PoissonLMM model have similar performance under both the null and alternative hypotheses. The main reason of the equivalent performance of these two tests is that the employed BNB model is based on the PoissonGamma compound distribution, which is similar to fit the PoissonLMM model. When testing γ>1 for paired designs, the Wald test of BNB is recommended since it has the highest power among all four tests. But when testing γ<1 for paired designs, either the LRT under the BNB model or the LRT under the PoissonLMM model is recommended. This unbalanced effect on power for the Wald test is related to selected transformation functions g(.), which was also reported in the article by Rettiganti and Nagaraja [13]. We also notice that there are some negative values of LRT statistics under the NBLMM model in our simulations. For these cases with negative values, the parameter space under the null hypothesis is not completely contained in the parameter space under the alternative hypothesis since the MLE estimates of the random effect under both hypotheses are not the same.
In almost all current studies that use a NB model for differential expression detection or power analysis, the dispersion parameter is assumed equal across conditions. We have proposed unequal dispersion parameters across conditions for power analysis [17]. Similarly, the proposed BNB model can be naturally extended to unequal dispersion parameters with both the LRT and Wald tests for paired designs. In addition, the proposed GLMM procedure can be applied to multiple factorial designs and allow multiple random effects. For these designs, the value or distribution of model parameters need to be prespecified so that the procedure can simulate data from a known GLMM model under both hypotheses. In addition, our proposed method can be used to estimate power at the data set level, where the distribution under the null hypothesis of the LRT or Wald test can be simulated for groups of genes with similar expression profile to maintain a proper false positive rate control.
Conclusions
Many methods on sample size calculation and power estimation have already been proposed for RNASeq data over last decade. However nearly all of them were developed for experiments with independent measurements. To overcome this limitation in current methods, we provide a framework for power analysis of RNASeq experiments with correlated measurements (e.g. repeated samples, time course, etc.). Our simulation based procedures provide proper control of false positive rate, and our novel GLMM procedure can be used for complex designs by allowing diverse correlation structures with both random effects and random residual errors.
Abbreviations
 LRT:

Likelihood ratio test
 BNB:

Bivariate negative binomial
 MLE:

Maximum likelihood estimation
 GLMM:

Generalized linear mixed effects model
 PCER:

Per comparison error rate
 TCGA:

The cancer genome atlas
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This research was partially supported by NIH grants 2P30CA01605840. The funding body did not play any roles in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
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All authors were involved in method development. LY generated the idea, performed analysis, and drafted the manuscript. GB and SF guided the research and revised the manuscript with equal contribution. All authors read and approved the final version of this manuscript.
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Correspondence to Lianbo Yu.
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Yu, L., Fernandez, S. & Brock, G. Power analysis for RNASeq differential expression studies using generalized linear mixed effects models. BMC Bioinformatics 21, 198 (2020). https://doi.org/10.1186/s1285902035417
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Keywords
 RNASeq
 Power analysis
 Bivariate negative binomial
 Generalized linear mixed effects Model