# Power and sample size analysis, Microarray data, RNAseq data

Maarten van Iterson[aut, cre] and Renee de Menezes [ctb]

#### 27 April 2020

#### Package

SSPA 2.28.0

modified: Tue Jan 28 20:29:29 2020 compiled: Mon Apr 27 22:33:45 2020

# 1 Introduction

Power and sample size analysis or sample size determination is concerned with the question of determining the minimum number of samples necessary to demonstrate the existence (or absence) of a difference between two or more populations of interest. The number of samples should be sufficient in that the statistical test will reject the null hypothesis, when there really exists a difference, with high probability or power.

Sample size determination for experiments involving high-dimensional data has several challenges as a multiple testing problem is involved, furthermore the occurrence of differentially and nondifferentialy expressed genes and that each gene individually has an effect size and a standard deviation leading to a distribution of effect sizes and standard deviations complicates the problem even further.

Power and sample size analysis for high-dimensional data was initiated by (Lee and Whitmore 2002). The multiple testing problem was controlled using the easy to apply family-wise error rate. Since controlling the family-wise error rate is too conservative for microarray data, methods were proposed that control the multiple testing problem using the false discovery rate (Pawitan et al. 2005; Liu and Hwang 2007; Tong and Zhao 2008). Recently, those methods were adapted for using pilot-data in order to obtain more realistic estimates of the power (Ferreira and Zwinderman 2006; Ruppert, Nettleton, and Hwang 2007; Midelfart and Bones 2008).

This vignette describes SSPA, a package implementing a general pilot data-based method for power and sample size determination for high-dimensional genomic data, such as those obtained from microarray or next-generation sequencing experiments. The method is based on the work of Ferreira and Zwinderman (Ferreira and Zwinderman 2006) and generalized as described by van Iterson et al. (Iterson et al. 2009, 2013).

By means of two simple commands (`pilotData()`, `sampleSize()`), a researcher can read in their data –a vector of test statistics– and compute the desired estimates. Other functions are provided that facilitate interpretation of results. Simulation data is used to show the general functionality and flexibility of the package and two interesting biological case study are shown.

# 2 Simulated data

This simulation study represents a two group microarray experiment with \(m = 5000\) features and \(N = 10\) samples per group. Let \(R_{ij}\) and \(S_{ij}\), \(i = 1, \ldots, m\), \(j = 1, \ldots, N\) be random variables where \(S_{ij} \sim N(\frac{-\mu_i}{2}, 1)\) and \(R_{ij} \sim N(\frac{\mu_i}{2}, 1)\) with the first \(\mu_{i} = 0\) for \(i = 1, \ldots, m_0\) and the remaining \(\mu_i\) for \(i = m_0 + 1, \ldots, m\) were drawn from a symmetric bi-triangular distribution as described by (Langaas, Lindqvist, and Ferkingstad 2005). A total of \(25\) data sets were simulated with the proportion of non-differentially expressed features fixed at \(\pi_0 = 0.8\). Additional technical noise is added to the model using \(X \sim \log(e^R + e^\epsilon)\) and \(Y \sim \log(e^S + e^\epsilon)\) with \(\epsilon \sim N(0, 0.1^2)\), so that the noise is positive and additive.

    library(SSPA)
    library(genefilter)
    set.seed(12345)
    m <- 5000
    J <- 10
    pi0 <- 0.8
    m0 <- as.integer(m * pi0)
    mu <- rbitri(m - m0,
                 a = log2(1.2),
                 b = log2(4),
                 m = log2(2))
    data <- simdat(
      mu,
      m = m,
      pi0 = pi0,
      J = J,
      noise = 0.01
    )
    statistics <-
      rowttests(data, factor(rep(c(0, 1), each = J)))$statistic

## 2.1 Deconvolution

The first step in doing the sample size and power analysis is creating a object of class `PilotData` which will contain all the necessary information needed for the following power and sample size analysis; a vector of test-statistics and the sample sizes used to compute them. A user-friendly interface for creating an object of `PilotData` is available as `pilotData()`.

Several ways of viewing the content of the PilotData object are possible either graphically or using a show-method by just typing the name of the created object of `PilotData`:

    pdD <- pilotData(
      statistics = statistics,
      samplesize = sqrt(1 / (1 / J + 1 / J)),
      distribution = "norm"
    )
    pdD

    ## Formal class 'PilotData' [package "SSPA"] with 5 slots
    ##   ..@ statistics  : num [1:5000] -1.98854 -0.00589 -1.26628 0.74421 -0.76088 ...
    ##   ..@ samplesize  : num 2.24
    ##   ..@ pvalues     : num [1:5000] 0.0468 0.9953 0.2054 0.4567 0.4467 ...
    ##   ..@ distribution: chr "norm"
    ##   ..@ args        : list()

    plot(pdD)

Figure 1: Exploratory plots of the pilot-data: Upper left panel shows a histogram of the test statistics, upper right panel a histogram of the p-values
Lower left panel shows a qq-plot for the test statistics using the user-defined null distributions. Lower right panel shows the sorted p-values against their ranks.

Now we can create an object of class `SampleSize` which will perform the estimation of the proportion of non-differentially expressed genes and the density of effect sizes. Several options are available see `?sampleSize`.

The density of effect size can be shown by a call to `plot()`. When there are both up- and down-regulated genes a bimodal density is observed.

    ssD <- sampleSize(pdD, control = list(from = -6, to = 6))
    ssD

    ## Formal class 'SampleSize' [package "SSPA"] with 10 slots
    ##   ..@ pi0         : num 0.781
    ##   ..@ lambda      : num [1:512] 0 0 0 0 0 0 0 0 0 0 ...
    ##   ..@ theta       : num [1:512] -6 -5.98 -5.95 -5.93 -5.91 ...
    ##   ..@ control     :List of 11
    ##   .. ..$ method    : chr "deconv"
    ##   .. ..$ pi0Method : chr "Langaas"
    ##   .. ..$ pi0       : num [1:90] 0.1 0.11 0.12 0.13 0.14 0.15 0.16 0.17 0.18 0.19 ...
    ##   .. ..$ adjust    : logi TRUE
    ##   .. ..$ a         : num 0.5
    ##   .. ..$ bandwith  : NULL
    ##   .. ..$ kernel    : chr "fan"
    ##   .. ..$ from      : num -6
    ##   .. ..$ to        : num 6
    ##   .. ..$ resolution: num 512
    ##   .. ..$ verbose   : logi FALSE
    ##   ..@ info        :List of 1
    ##   .. ..$ pi0: num 0.767
    ##   ..@ statistics  : num [1:5000] -1.98854 -0.00589 -1.26628 0.74421 -0.76088 ...
    ##   ..@ samplesize  : num 2.24
    ##   ..@ pvalues     : num [1:5000] 0.0468 0.9953 0.2054 0.4567 0.4467 ...
    ##   ..@ distribution: chr "norm"
    ##   ..@ args        : list()

    plot(
      ssD,
      panel = function(x, y, ...)
      {
        panel.xyplot(x, y)
        panel.curve(dbitri(x),
                    lwd = 2,
                    lty = 2,
                    n = 500)
      },
      ylim = c(0, 0.6)
    )

Figure 2: Estimated density of effect sizes: True density of effect sizes, the bitriangular distribution, and estimated density of effect sizes in blue

Estimating the average power for other sample sizes then that of the pilot-data can be performed with the `predictpower()`-function. The user can also give the desired false discovery rate level.

    Jpred <- seq(10, 20, by = 2)
    N <- sqrt(Jpred / 2)
    pwrD <- predictpower(ssD, samplesizes = N, alpha = 0.05)
    matplot(
      Jpred,
      pwrD,
      type = "b",
      pch = 16,
      ylim = c(0, 1),
      ylab = "predicted power",
      xlab = "sample size (per group)"
    )
    grid()

Figure 3: Predicted power curve: For sample sizes from 10 to 20 the power is predicted based on the information obtained from the pilot-data

The deconvolution estimator of the density of effect sizes is only applicable for two-group comparisons when the test statistics is (approximate) normally distributed. Note that in this situation we use the effective sample size. In all other cases e.g. two group comparison using the \(t\) statistics with the Conjugate Gradient estimator for the density of effect sizes the total sample size is used.

## 2.2 Conjugate Gradient

    pdC <- pilotData(
      statistics = statistics,
      samplesize = sqrt(2 * J),
      distribution = "t",
      df = 2 * J - 2
    )
    ssC <- sampleSize(pdC,
                      method = "congrad",
                      control = list(
                        from = -6,
                        to = 6,
                        resolution = 250
                      ))
    plot(
      ssC,
      panel = function(x, y, ...)
      {
        panel.xyplot(x, y)
        panel.curve(2 * dbitri(2 * x),
                    lwd = 2,
                    lty = 2,
                    n = 500)
      },
      xlim = c(-2, 2),
      ylim = c(0, 1.2)
    )

Figure 4: Conjugate gradient method: Estimated density of effect sizes using the conjugate gradient method

## 2.3 Tikhonov regularization

    ssT <- sampleSize(
      pdC,
      method = "tikhonov",
      control = list(
        resolution = 250,
        scale = "pdfstat",
        lambda = 10 ^ seq(-10, 10, length = 50),
        verbose = FALSE,
        modelselection = "lcurve"
      )
    )
    plot(
      ssT,
      panel = function(x, y, ...)
      {
        panel.xyplot(x, y, type = "b")
        panel.curve(2 * dbitri(2 * x),
                    lwd = 2,
                    lty = 2,
                    n = 500)
      },
      xlim = c(-2, 2),
      ylim = c(0, 1.2)
    )

Figure 5: Tikohonov regularization: Estimated density of effect sizes using the Tikohonov regularization

# 3 Case Studies:

## 3.1 Nutrigenomics microarray data

In this first example a set consisted of Affymetrix array data derived from a nutrigenomics experiment in which weak, intermediate and strong PPAR\(\alpha\) agonists were administered to wild-type and PPAR\(\alpha\)-null mice is used. The power and sample size analysis confirms the hierarchy of PPAR\(\alpha\)-activating compounds previously reported and the general idea that larger effect sizes positively contribute to the average power of the experiment.

PPAR\(\alpha\) is a transcription factor that is activated upon binding by a variety of agonists, both of synthetic and natural origin. In this experiment the outcome of specific PPAR\(\alpha\) activation on murine small intestinal gene expression was examined using Affymetrix GeneChip Mouse 430 2.0 arrays. PPAR\(\alpha\) was activated by several PPAR\(\alpha\)-agonists that differed in activating potency. Data of three agonists were used, namely Wy14,643, fenofibrate and trilinolenin (C18:3). The first two compounds belong to the fibrate class of drugs that are widely prescribed to treat dyslipidemia, whereas trilinolenin is an agonist frequently found in the human diet. For intestinal PPAR\(\alpha\), Wy14,643 is the most potent agonist followed by C18:3 and fenofibrate. Since time of exposure also affects the effect size, intestines were collected 6 hrs (all three agonists) or 5 days (Wy14,643 and fenofibrate only) after exposure. Expression estimates of probesets were obtained by GC-robust multi-array (GCRMA) analysis, employing the empirical Bayes approach for background adjustment, followed by quantile normalization and summarization. For each compound and exposure time, lists of moderated t-test statistics were computed, using the empirical Bayes linear regression model as implemented in limma, for each contrast representing the effect of compound in PPAR\(\alpha\)-null mice compared to wild-type mice. For more details see (Iterson et al. 2009).

    library(lattice)
    data(Nutrigenomics)
    str(Nutrigenomics)

    ## 'data.frame':    16540 obs. of  5 variables:
    ##  $ wy5d  : num  2 1.22 1.19 -1.14 1 0.86 4 -1.83 -2.64 3.04 ...
    ##  $ feno5d: num  2 -1.03 -1.11 0.33 0.22 0.22 0.24 0.36 1.73 0.54 ...
    ##  $ X1836h: num  2.5 -2.3 -0.56 0.06 -0.89 -0.09 2 -2.12 -1.47 -1.69 ...
    ##  $ wy6h  : num  2 1.12 0.21 -0.68 -1.06 -0.4 1.82 -1.71 1.2 1.03 ...
    ##  $ feno6h: num  2.22 0.76 -0.37 -0.07 -1.9 ...

    pd <- apply(Nutrigenomics, 2,
                function(x)
                  pilotData(
                    statistics = x[-1],
                    samplesize = sqrt(x[1]),
                    distribution = "norm"
                  ))
    ss <- lapply(pd,
                 sampleSize,
                 control = list(
                   pi0Method = "Storey",
                   a = 0,
                   resolution = 2 ^ 10,
                   verbose = FALSE
                 ))

    ##ss <- lapply(pd, sampleSize,
    ##             method = "congrad",
    ##             control=list(verbose=FALSE, resolution=2^10, from=-10, to=10))

    compounds <-
      c("Wy14,643",
        "fenofibrate",
        "trilinolenin (C18:3)",
        "Wy14,643",
        "fenofibrate")
    exposure <- c("5 Days", "6 Hours")

    effectsize <-
      data.frame(
        exposure = factor(rep(rep(exposure, c(
          2, 3
        )), each = 1024)),
        compound = factor(rep(compounds, each = 1024)),
        lambda = as.vector(sapply(ss,
                                  function(x)
                                    x@lambda)),
        theta = as.vector(sapply(ss,
                                 function(x)
                                   x@theta))
      )

    print(
      xyplot(
        lambda ~ theta | exposure,
        group = compound,
        data = effectsize,
        type = c("g", "l"),
        layout = c(1, 2),
        lwd = 2,
        xlab = "effect size",
        ylab = "",
        auto.key = list(
          columns = 3,
          lines = TRUE,
          points = FALSE,
          cex = 0.7
        )
      )
    )

Figure 6: Nutrigenomic example: Estimated density of effect sizes for the five treatment exposure conditions

    samplesize <- seq(2, 8)
    averagepower <- data.frame(
      power = as.vector(sapply(ss,
                               function(x)
                                 as.numeric(
                                   predictpower(x, samplesize = sqrt(samplesize))
                                 ))),
      exposure = factor(rep(rep(exposure, c(
        2, 3
      )), each = length(samplesize))),
      compound = factor(rep(compounds, each = length(samplesize))),
      samplesize = rep(2 * samplesize, 5)
    )

    print(
      xyplot(
        power ~ samplesize |
          exposure,
        group = compound,
        data = averagepower,
        type = c("g", "b"),
        layout = c(1, 2),
        lwd = 2,
        pch = 16,
        xlab = "sample size (per group)",
        ylab = "",
        auto.key = list(
          columns = 3,
          lines = TRUE,
          points = FALSE,
          cex = 0.7
        )
      )
    )

Figure 7: Nutrigenomic example: Predicted power curves for the five treatment exposure conditions

## 3.2 deepSAGE of wild-type vs Dclk1 transgenic mice

In this example we will show how our method can be applied to count data of an RNA-seq experiment. We will use the data described by (Hoen et al. 2008) comparing gene expression profiles in the hippocampi of transgenic \(\delta\)C-doublecortin-like kinase mice with that of wild-type mice. Data is available from GEO (GSE10782).

and analyzed using the generalized linear model approach implemented in edgeR. A tag-wise dispersion parameter was estimated using the Cox-Reid adjusted profile likelihood approach for generalized linear models as implemented in edgeR. Using a treatment contrast, we tested per tag if there was a genotype effect using the likelihood ratio statistic. This test statistic follow asymptotically a \(\chi^2\) distribution with one degree of freedom.

    ##files contains the full path and file names of each sample
    targets <- data.frame(files = files,
                          group = rep(c("DCLK", "WT"), 4),
                          description = rep(
                            c(
                              "transgenic (Dclk1) mouse hippocampus",
                              "wild-type mouse hippocampus"
                            ),
                            4
                          ))
    d <- readDGE(targets) ##reading the data
    ##filter out low read counts
    cpm.d <- cpm(d)
    d <- d[rowSums(cpm.d > 1) >= 4,]

    design <- model.matrix( ~ group, data = d$samples)
    ##estimate dispersion
    disp <- estimateGLMCommonDisp(d, design)
    disp <- estimateGLMTagwiseDisp(disp, design)
    ##fit model
    glmfit.hoen <-
      glmFit(d, design, dispersion = disp$tagwise.dispersion)
    ##perform likelihood ratio test
    lrt.hoen <- glmLRT(glmfit.hoen)
    ##extract results
    tbl <- topTags(lrt.hoen, n = nrow(d))[[1]]
    statistics <- tbl$LR

    library(lattice)
    data(deepSAGE)
    str(deepSAGE)

    ##  num [1:44882] 133.5 93.8 89.5 88.8 87.4 ...

    pd <- pilotData(
      statistics = deepSAGE,
      samplesize = 8,
      distribution = "chisq",
      df = 1
    )
    ss <- sampleSize(pd,
                     method = "congrad",
                     control = list(
                       trim = c(0, 0.98),
                       symmetric = FALSE,
                       from = 0,
                       to = 10
                     ))
    pwr <- predictpower(ss, samplesize = c(8, 16, 32))
    op <- par(mfcol = c(2, 1), mar = c(5, 4, 1, 1))
    plot(ss@theta,
         ss@lambda,
         xlab = "effect size",
         ylab = "",
         type = "l")
    plot(
      c(8, 16, 32),
      pwr,
      xlab = "sample size",
      ylab = "power",
      type = "b",
      ylim = c(0, 1)
    )
    grid(col = 1)

Figure 8: Deep SAGE example: Estimated density of effect size and predicted power curve

    par(op)

# 4 Session Info

Here is the output of `sessionInfo()` on the system on which this document was compiled:

    ## R version 4.0.0 (2020-04-24)
    ## Platform: x86_64-w64-mingw32/x64 (64-bit)
    ## Running under: Windows Server 2012 R2 x64 (build 9600)
    ##
    ## Matrix products: default
    ##
    ## locale:
    ## [1] LC_COLLATE=C
    ## [2] LC_CTYPE=English_United States.1252
    ## [3] LC_MONETARY=English_United States.1252
    ## [4] LC_NUMERIC=C
    ## [5] LC_TIME=English_United States.1252
    ##
    ## attached base packages:
    ## [1] stats     graphics  grDevices utils     datasets  methods   base
    ##
    ## other attached packages:
    ## [1] genefilter_1.70.0 lattice_0.20-41   SSPA_2.28.0       BiocStyle_2.16.0
    ##
    ## loaded via a namespace (and not attached):
    ##  [1] qvalue_2.20.0        tidyselect_1.0.0     xfun_0.13
    ##  [4] purrr_0.3.4          reshape2_1.4.4       splines_4.0.0
    ##  [7] colorspace_1.4-1     vctrs_0.2.4          htmltools_0.4.0
    ## [10] stats4_4.0.0         yaml_2.2.1           survival_3.1-12
    ## [13] blob_1.2.1           XML_3.99-0.3         rlang_0.4.5
    ## [16] pillar_1.4.3         glue_1.4.0           DBI_1.1.0
    ## [19] BiocGenerics_0.34.0  bit64_0.9-7          lifecycle_0.2.0
    ## [22] plyr_1.8.6           stringr_1.4.0        munsell_0.5.0
    ## [25] gtable_0.3.0         memoise_1.1.0        evaluate_0.14
    ## [28] Biobase_2.48.0       knitr_1.28           IRanges_2.22.0
    ## [31] parallel_4.0.0       AnnotationDbi_1.50.0 highr_0.8
    ## [34] Rcpp_1.0.4.6         xtable_1.8-4         scales_1.1.0
    ## [37] BiocManager_1.30.10  limma_3.44.0         S4Vectors_0.26.0
    ## [40] magick_2.3           annotate_1.66.0      bit_1.1-15.2
    ## [43] ggplot2_3.3.0        digest_0.6.25        stringi_1.4.6
    ## [46] bookdown_0.18        dplyr_0.8.5          grid_4.0.0
    ## [49] tools_4.0.0          bitops_1.0-6         magrittr_1.5
    ## [52] RCurl_1.98-1.2       tibble_3.0.1         RSQLite_2.2.0
    ## [55] crayon_1.3.4         pkgconfig_2.0.3      Matrix_1.2-18
    ## [58] ellipsis_0.3.0       assertthat_0.2.1     rmarkdown_2.1
    ## [61] R6_2.4.1             compiler_4.0.0

# References

Ferreira, J. A., and A. Zwinderman. 2006. “Approximate Sample Size Calculations with Microarray Data: An Illustration.” Statistical Applications in Genetics and Molecular Biology 5 (1): 1.

Hoen, P. A. C. ’t, Y. Ariyurek, H. H. Thygesen, E. Vreugdenhil, R. H. A. M. Vossen, R. X. de Menezes, J. M. Boer, G. B. van Ommen, and J. T. den Dunnen. 2008. “Deep Sequencing-Based Expression Analysis Shows Major Advances in Robustness, Resolution and Inter-Lab Portability over Five Microarray Platforms.” Nucleic Acids Research, 1–11.

Iterson, M. van, P. A. C. ’t Hoen, P. Pedotti, G. J. E. J. Hooiveld, J. T. den Dunnen, G. J. B. van Ommen, J. M. Boer, and R. X. de Menezes. 2009. “Relative Power and Sample Size Analysis on Gene Expression Profiling Data.” BMC Genomics 10 (3): 439–49.

Iterson, M. van, M. A. van de Wiel, J. M. Boer, and R. X. de Menezes. 2013. “Power and Sample Size Calculations for High-Dimensional Genomic Data.” Statistical Applications in Genetics and Moleculair Biology 12 (4): 449–67.

Langaas, M., B. H. Lindqvist, and E. Ferkingstad. 2005. “Estimating the Proportion of True Null Hypotheses, with Application to Dna Microarray Data.” Journal of the Royal Statistical Society Series B 67 (4): 555–72.

Lee, M. L. T., and G. A. Whitmore. 2002. “Power and Sample Size for Dna Microarray Studies.” Statistics in Medicine 21: 3543–70.

Liu, P., and J. T. G. Hwang. 2007. “Quick Calculation for Sample Size While Controlling False Discovery Rate with Application to Microarrays.” Bioinformatics 26 (6): 739–46.

Midelfart, T. S. Jand H., and A. M. Bones. 2008. “A Mixture Model Approach to Sample Size Estimation in Two-Sample Comparative Microarray Experiments.” BMC Bioinformatics 9 (117): 1.

Pawitan, Y., S. Michiels, S. Koscielny, A. Gusnanto, and A. Ploner. 2005. “False Discovery Rate, Sensitivity and Sample Size for Microarray Studies.” Bioinformatics 21 (13): 3017–24.

Ruppert, D., D. Nettleton, and J. T. G. Hwang. 2007. “Exploring the Information in P-Values for the Analysis and Planning of Multiple-Test Experiments.” Biometrics 63 (2): 483–95.

Tong, T., and H. Zhao. 2008. “Practical Guidelines for Assessing Power and False Discovery Rate for Fixed Sample Size in Microarray Experiments.” Statistics in Medicine 27: 1960–72.