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Hierarchicell: an Rpackage for estimating power for tests of differential expression with singlecell data
BMC Genomics volume 22, Article number: 319 (2021)
Abstract
Background
Study design is a critical aspect of any experiment, and sample size calculations for statistical power that are consistent with that study design are central to robust and reproducible results. However, the existing power calculators for tests of differential expression in singlecell RNAseq data focus on the total number of cells and not the number of independent experimental units, the true unit of interest for power. Thus, current methods grossly overestimate the power.
Results
Hierarchicell is the first singlecell power calculator to explicitly simulate and account for the hierarchical correlation structure (i.e., within sample correlation) that exists in singlecell RNAseq data. Hierarchicell, an Rpackage available on GitHub, estimates the within sample correlation structure from real data to simulate hierarchical singlecell RNAseq data and estimate power for tests of differential expression. This multistage approach models gene dropout rates, intraindividual dispersion, interindividual variation, variable or fixed number of cells per individual, and the correlation among cells within an individual. Without modeling the within sample correlation structure and without properly accounting for the correlation in downstream analysis, we demonstrate that estimates of power are falsely inflated. Hierarchicell can be used to estimate power for binary and continuous phenotypes based on userspecified number of independent experimental units (e.g., individuals) and cells within the experimental unit.
Conclusions
Hierarchicell is a userfriendly Rpackage that provides accurate estimates of power for testing hypotheses of differential expression in singlecell RNAseq data. This Rpackage represents an important addition to singlecell RNA analytic tools and will help researchers design experiments with appropriate and accurate power, increasing discovery and improving robustness and reproducibility.
Background
Robust and reproducible science depends on the quality of the experimental design. High quality experimental design revolves around focused research questions or hypotheses, appropriate and valid measures of the central variables related to these hypotheses, statistically sound analysis plans, and properly computed power analysis [1]. While power analyses for genetic association studies and bulk RNAseq approaches are wellestablished [2,3,4,5,6], such analyses remain a challenge in singlecell RNAseq studies due to intrasample correlation inherent in these data [7]. Such within sample correlations exist because cells from the same individual share a common genetic and environmental background that often leads to greater similarity in gene expression among cells in the same sample. Therefore, gene expression measures among cells from the same sample have a hierarchical correlation structure where cells nested within an individual are not independent units. At present, the correlative (hierarchical) nature of these data is often neglected, in both power analyses and tests of hypotheses (e.g., differential expression) [7]. This was recently highlighted in a valuable paper by Andrews et al. which states that “current singlecell differential expression tests treat each individual cell as a biological replicate and cannot account for shared genetic backgrounds or disease state”. [8] Ignoring the hierarchal nature of singlecell RNAseq data leads to studies that are under powered and inappropriately analyzed [7], leading to incorrect inference, poor reproducibility, and financial investments in those errors. A contributor to these flawed practices is the void of singlecell specific methods and literature that properly account for this hierarchical structure. However, just as Andrews et al. pointed out, we – too  expect that, “as scRNAseq is applied to larger cohorts and comparison studies, [there will be] further developments that lead to more accurate statistical models for more complex experimental designs.” An excellent starting place for more accurate statistical models are accurate power calculations for improved study design.
Besides the classic, closed form, normal theory power calculations (e.g., ANOVA) that make too many overly simplistic assumptions (e.g., normality, independence), the power calculators for testing singlecell RNA differential expression all simulate cells independently, without the withinsubject correlation structure [9,10,11]. Previously, we documented that in tests of differential expression in singlecell RNAseq data one needs to account for the within experimental unit (e.g., individual) and showed that mixedeffects models with subject/individual as a random effect is a practical and statistically sound approach for these hypotheses [7]. Here, we present an Rpackage, Hierarchicell, with two purposes: 1) it is a simulator of hierarchical singlecell RNAseq data, and 2) it computes power estimates using a mixedeffects models for testing hypotheses of differential gene expression in singlecell RNAseq data. Hierarchicell simulates singlecell RNAseq data with a hierarchical structure that closely resembles that of real data and can be used by researchers to make informed choices on experimental design while balancing the tradeoff between cost and power. Our Rpackage is user friendly and flexible to a variety of scenarios. It incorporates estimates from real data [12] or allows users to input data (e.g., either Fluidigm C1 or 10x Chromium technology, user’s own pilot data) to obtain highly translatable and accurate estimates of power tailored to their technology. Within a wellcharacterized set of parameters that are modeled from either a userdefined or the default singlecell RNAseq data, the tool provides users with estimates of power relative to a given foldchange, significance threshold, number of independent samples, and number of cells per independent sample. In addition, the calculator allows for the simulation of either continuous or binary phenotypes of interest. For binary casecontrol analyses, the user specifies the foldchange they desire to detect. For continuous phenotypes, the user specifies the mean and standard deviation of the phenotype and the degree of correlation with expression the user desires to detect with significance. Currently, most singlecell power calculators only provide estimates for the required number of cells rather than the required number of independent experimental units (e.g. individuals) or are not designed for computing power to detect differences in expression [13,14,15]. Other power calculations for singlecell RNAseq are based on bulk RNAseq methods to estimate the required number of samples [2, 3]. Estimating power for a singlecell RNAseq study using bulk RNAseq power calculators is a reasonable solution, but will underestimate the study’s power by not incorporating the additional power gained by sequencing numerous cells per individual. This tool provides a valuable resource in an area of critical need for researchers looking to optimize their study’s power and experimental design relative to the hierarchical nature that exists in all singlecell data.
Implementation
Overview of the Hierarchicell simulation engine
A stepbystep overview of the simulation procedure is provided with Rcode examples and detailed explanations in Hierarchicell’s accompanying vignette. We encourage users to review this vignette (available on GitHub and in the Supplementary Materials) before beginning to work with Hierarchicell. The singlecell data in that example are used to estimate default simulation parameters for our simulation engine. These data were downloaded from the public accession number EMTAB5061 [12]. These data were sequenced using the SmartSeq2 protocol and they include sequence data from 3514 cells from 10 different individuals [12]. Genes were previously normalized to account for the differences in library size [12]. After filtering down to high quality alpha cells, our dataset contained gene expression values for 22,983 genes and 886 cells (across 10 individuals). This dataset is included as part of the Rpackage for a number of reasons. Primarily, these data demonstrate the general intra and interindividual correlation patterns seen across a variety of singlecell data of different cell types generated by different platforms [7]. In addition, these data are not too large, allowing for the rapid estimation of simulation parameters while also minimizing the size of the Rpackage.
The simulation procedure was designed to simulate independent genes in a way that approximates the hierarchical structure of real data by empirically estimating the range of parameters (i.e., grand mean of the transcriptpermillion (TPM) values, within sample variance, between sample variance, relationship between the grand mean and dispersion, dropout) that define the observed distribution of TPM values for a gene. To estimate these parameters, genes were pruned to a set of uncorrelated genes that captured the most representative patterns of detectable TPM values, without the resulting parameter estimates being primarily driven by dropout. Specifically, genes were sequentially sampled one at a time and any other gene having transcript abundances correlated (Spearman’s correlation coefficient > 0.25) with the gene were removed. To estimate the grand means independently from the hierarchical correlation structure, the grand means were estimated by sampling one cell from each individual and computing the mean TPM value 1000 times. The mean of each of those means was used to approximate the grand mean. To approximate the variance of the withinsample means (interindividual variance), the means of all nonzero TPM values were computed across all cells within each individual and the variance between those values was subsequently computed. To estimate the withinsample dispersion values, the nonzero TPM values were first used to compute a withinsample variance and withinsample mean. Consistent with the classical definition of the Negative Binomial distribution’s dispersion parameter, the withinsample dispersion parameter was then computed as:
where α_{ij} represents the dispersion parameter, μ_{ij} represents the withinsample mean, and \( {\sigma}_{ij}^2 \) represents the withinsample variance for gene i and individual j. The grand means and variances were computed empirically from the TPM values previously reported in six different cell types across three different singlecell studies [12, 16, 17]. Once consistent patterns were identified across cell types, alpha cells from the pancreas dataset, were used as the model data for our simulation. A gamma distribution was fit to the global mean of the TPM values of each gene using maximumlikelihood estimation. For each independently simulated gene i, a random value was sampled from this gamma distribution to obtain a grand mean, μ_{i}. The variance of the withinsample means (interindividual variance) was modeled as a linear function of the grand means, f_{1}(μ_{i}) and the withinsample dispersion (intraindividual variance) was estimated as a logarithmic function of the withinsample means, f_{2}(μ_{i}), and the probability of dropout was estimated independently as a bounded gamma distribution (Fig. 1). Using a normal distribution with an expected value of zero and a variance computed by the first linear relationship, f_{1}(μ_{i}), a difference in means was drawn for each of the individuals j in the simulation. This difference was summed with the grand mean to obtain an individual mean, μ_{ij}. Three different methods were used to simulate the number of cells per individual. To simulate scenarios where each of the individuals had the exact same number of cells, the number of cells desired for each individual was fixed at a constant value. In order to simulate scenarios where the number of cells per individual demonstrated slight imbalance, a Poisson distribution with a λ equal to the expected number of cells desired for each individual was then used to obtain the count of cells for each individual. To simulate scenarios where the number of cells per individual demonstrated greater imbalance, the number of cells per individual were modeled as a Negative Binomial random variable with a mean equal to the expected number of cells and a dispersion parameter of one. For each gene i and cell k assigned to an individual j, a read count value, Y_{ijk}, was drawn from a Negative Binomial distribution with an expected value equal to the individual’s assigned read count value, μ_{ij}, and a dispersion parameter, α_{ij}, computed by the logarithmic function of the grand mean f_{2}(μ_{i}).
Overview of Hierarchicell power calculations
To compute power, transcriptspermillion (TPM) values were simulated for each gene with the userspecified foldchange or ρ parameter (Fig. 1). Foldchange should be specified where users are interested in computing power for two distinct groups. The ρ parameter, which represents the degree of correlation between gene expression and a simulated continuous phenotype, should be specified where users are interested in computing power for association analysis with a continuous trait. Here, foldchange is a constant that was multiplied by the global mean gene expression values to spike the expression of those genes in the desired treatment group. The direction of the foldchange was simulated with a Bernoulli distribution with a probability of 0.5 to allow the direction of effect to vary equally between groups.
We applied a twopart hurdle model with a random effect for individual as directed in MAST’s reference manual (7,18). Specifically, a log(x + 1) transformation of the data was applied and the hurdle model computed to find genes exhibiting differences in expression. Using their same notation, the addition of random effects for differences among persons is as follows:
where Y_{ig} is the expression level for gene g and cell i, Z_{ig} is an indicator for whether gene g is expressed in cell i, X_{i} contains the predictor variables for each cell i, and W_{i} is the design matrix for the random effects of each cell i belonging to each individual j (i.e., the random complement to the fixed X_{i}). β_{g} represents the vector of fixedeffects regression coefficients and γ_{j} represents the vector of random effects (i.e., the random complement to the fixed β_{g}). γ_{j} is distributed normally with a mean of zero and variance \( {\sigma}_g^2 \). To obtain a single result for each gene, likelihood ratio or Wald test results from each of the two components are summed and the corresponding degrees of freedom for each component are added. These tests have asymptotic χ^{2} null distributions; these statistics can be summed and remain asymptotically χ^{2} because Z_{g} and Y_{g} are defined conditionally independent for each gene. When summed together, these tests provide a single test for the twopart hurdle model. Our package also offers the ability to compute type 1 error rates (and thereby power) for a variety of different singlecell analysis approaches. New methods that properly handle within sample correlation will be integrated as they become available.
Software implementation
All simulations and data were compiled in RStudio using R3.6.2 and is freely available on GitHub. The supplementing dataset that is included to run the Rpackage without user input data was significantly downsized by removing all of the genes correlated with a Spearman’s correlation coefficient > 0.25. This filtering is one of the first steps in our simulation procedure and doing so greatly reduced the size of the source package to 475 KB as well as the data structures held in memory during use. Currently, the simulation typically completes in less than 5 seconds, depending on user specifications. The simulationbased power calculations, however, can take much longer depending on the model that is used. For the recommended twopart hurdle mixed model (MAST with a random effect for individual), this can range anywhere from 1 to 20 min per simulationbased estimate of power for a given foldchange on a 64bit Operating system with 8 CPUs and 16 GB of RAM. We note that these run times are heavily dependent on the number of genes, the sample size, the number of cells per individual specified, and the number of CPUs available.
Results and discussion
Previously, we demonstrated that our simulation recapitulates the most important aspects of singlecell gene expression data, particularly the hierarchical structure of singlecell RNAseq data which is rarely accounted for in differential expression analysis [7]. We also applied our simulation engine to demonstrate that mixed models are a statistically sound method that accounts for the within sample correlation and has appropriate type 1 error control without sacrificing power [7]. In addition, we provided power estimates for binary outcomes across a range of experimental conditions to assist researchers in designing appropriately powered studies [7].
We previously reported power calculations for tests of differential expression in singlecell RNAseq studies for binary phenotypes (i.e., case/control treatment groups) [7]. Hierarchicell now also allows users to estimate power for detecting associations between continuous traits and singlecell gene expression (Fig. 2). The simulated expression data can be computed over a range of correlations with the magnitude of expression in each individual’s cells, while accounting for the hierarchical structure of these data.
As expected, increasing the number of independent experimental units (e.g., individuals) in a study is the best way to increase power to detect true differences between traits measured at the individual and not individual cell level (Fig. 3a). Power calculations for binary phenotypes consisting of 10 individuals per treatment group reveal that there are only marginal gains in power when more than 100 cells per individual are sampled for a particular analysis unit (Fig. 3b). We also note that methods that do not account for within person correlation grossly overestimate power. For example, when estimating power with an approach that estimates the power for cells as independent units (assuming a type 1 error rate of α = 0.05, a fold change of 1.3, 10 individuals per treatment and 100 cells per individual), the power is overestimated as 0.93 instead of 0.71 when appropriately accounting for the within person correlation. Power calculations for continuous phenotypes, with the same sample sizes and constant withinperson correlations among cells, demonstrate even smaller gains in power when more than 100 cells per individual are sampled for a particular analysis unit (Fig. 3c). The gains in power from sampling more cells per individual will decrease as the numbers of independent experimental units increase (Fig. 3d). This is true for both types of analyses. As the degree of correlation among cells within a person decreases and approaches zero, rarely observed, the value tradeoff between independent experimental units and individual cells will vary. Further, we note that if the celltype of interest has much more or much less zeroinflation (i.e., less information), then the gains in power from sampling more cells may be greater or smaller, respectively. This is why estimating the data structure of the cell types of interest from preliminary data is a critically important feature of our Hierarchicell package. To consistently identify foldchange differences of at least 1.2 as statistically significant (power > 0.80), we approximate that researchers will need a minimum of 40 samples per group and 100 cells per sample in a classical case/control design. To consistently identify genes correlated with a correlation coefficient of 0.4 with a phenotype (power > 0.80), we approximate that researchers will need a minimum of 100 samples and 100 cells per sample.
As experiments get larger, computational time will increase. Future work will parallelize the code. To more rapidly close in on plausible sample size options, a researcher can apply the aggregate (“pseudobulk) methods power estimates and as one approaches feasible design shift to refining the estimates using the twopart hurdle mixed model employed here. However, it is important to do this refining step given the differences between these two approaches and the types of scenarios where aggregation methods will be underpowered [7, 18].
Future iterations of this package will incorporate any novel singlecell RNAseq differential expression methods that properly account for the within sample correlation. In addition, we will parallelize the code and improve the speed of software by building components of the software in other languages (such as C++ via rcpp), and/or storing results of a large number of scenarios for quick and easy access to the necessary information. Future developments would be to incorporate the relationship between power and the variance explained by an effect, not simply foldchange between treatments. In real data, the variances explained by an effect fluctuate greatly among genes and cell types. While the simulated expression data herein have variances that are modeled after real data and are allowed to fluctuate by genes, simulating a direct relationship between the variance and an effect will be a meaningful addition to this work.
Conclusions
To date, none of the primary power calculation methods are directly applicable for differential expression analysis with singlecell RNAseq data. Here, we present an Rpackage, Hierarchicell, with two purposes: 1) simulation of hierarchical singlecell RNAseq data, and 2) computation of power estimates using a mixedeffects models for testing hypotheses of differential gene expression in singlecell RNAseq data. Hierarchicell allows for a range of inputs and parameter settings and even the evaluation of various singlecell specific methods, but encourages using linear mixed models with individual as a random effect for both binary and continuous outcomes, as implemented in MAST [7, 19]. We recommend these mixed effects models because they retain appropriate type 1 error rates while maintaining power. Proper calculation of statistical power coupled with proper analysis methods that account for the correlation among cells from the same individual will increase robustness and reproducibility of singlecell studies, thereby reducing the cost while accelerating the rate of scientific discovery.
Availability and requirements

Project name: hierarchicell

Project home page: https://github.com/kdzimm/hierarchicell

Operating system(s): Linux, Mac, and PC

Programming language: R

Other requirements: No

License: CC0

Any restrictions to use by nonacademics: No
Availability of data and materials
The data used for the simulated data herein and in the longform documentation (R vignette) are available under the accession number EMTAB5061 at https://www.ebi.ac.uk/arrayexpress/experiments/EMTAB5061/. The Rpackage is freely available on GitHub at https://github.com/kdzimm/hierarchicell (DOI: https://doi.org/10.5281/zenodo.4608738).
Abbreviations
 RNASeq:

Sequencing technique which uses nextgeneration sequencing to reveal the presence and quantity of RNA
 MAST:

Modelbased Analysis of Singlecell Transcriptomics
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Acknowledgments
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Funding
This work was supported by The Center for Public Health Genomics and grant U01 NS036695 (CoPI Langefeld) from NIH, Department of Defense W81XWH2010686, and by the Cancer Center Support Grant from the National Cancer Institute to the Comprehensive Cancer Center of Wake Forest Baptist Medical Center (P30CA012197).
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CDL and KDZ conceived the study. KDZ constructed the Rpackage and implemented all simulations and analyses with guidance from CDL. KDZ wrote the original draft and reviewed and edited it with CDL. All authors approved the final version of the manuscript.
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Zimmerman, K.D., Langefeld, C.D. Hierarchicell: an Rpackage for estimating power for tests of differential expression with singlecell data. BMC Genomics 22, 319 (2021). https://doi.org/10.1186/s1286402107635w
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Keywords
 Hierarchical data
 Singlecell
 RNAsequencing
 Power calculator
 Simulation
 Rpackage
 Mixedeffects models