# Using radEmu on clustered data

#### Sarah Teichman and Amy Willis

#### 2026-03-20

First, we will install `radEmu`, if we haven’t already.

    # if (!require("remotes", quietly = TRUE))
    #     install.packages("remotes")
    #
    # remotes::install_github("statdivlab/radEmu")

Next, we can load `radEmu` as well as the `tidyverse` package suite.

    library(magrittr)
    library(dplyr)
    library(ggplot2)
    library(stringr)
    library(radEmu)

## Introduction

In this vignette we will explore the use of `radEmu` with clustered data. Data with cluster dependence is a common phenomenon in microbiome studies. This type of dependence can come from experimental factors such as shared cages or tanks for study animals. It can also arise from repeated measurements in longitudinal studies.

Sometimes people deal with clustered data via random effects. You might have seen the syntax `+ (1 | cluster)`. We (the `radEmu` developers) don’t want to make strong assumptions (such as the random effects being normally distributed), so we handle cluster dependence using a GEE framework. We like this approach because it’s robust to many forms of model misspecification, and for this reason, we find it superior to random effects.

When dependence is not accounted for, statistical inference in `radEmu` (and most statistical methods!) assume that all samples are independent. If you have cluster dependence but assume independence, you’ll have anti-conservative inference (i.e., p-values that are smaller than they should be). Therefore, we strongly recommend adjusting for cluster dependence if it arose in your sample collection!

Note that cluster dependence won’t change your estimates (\(\hat{\beta}_j\)’s), but it will (most likely) change your p-values.

Luckily, we have tools to account for cluster dependence implemented in `radEmu`! This argument is only implemented from `radEmu` v1.2.0.0 forward, so if you are having trouble using the `cluster` argument, check that you reinstalled `radEmu` recently.

TLDR; the basic syntax is as follows. `my_clusters` is a vector of length \(n\) (your number of samples), with observations from the same cluster having the same value in `my_clusters` (e.g. `my_clusters = c(1, 1, 2, 2, 3, 4)`).

    emuFit(formula = ~ covariate,
           data = my_data_frame,
           Y = my_microbial_abundances,
           cluster = my_clusters)

Fun fact! We implemented this functionality because of user requests. Therefore, if there’s something that you’d like to see that you don’t see, let us know and we’ll see what we can do!

## Generating data with cluster dependence

To start, let’s generate a toy example (10 categories, 60 samples) in which there is cluster dependence within our data. The way we simulate data isn’t important; it’s just an illustration. radEmu can handle more taxa and samples, we just did this so that the vignette builds quickly.

    J <- 10; n <- 60
    # generate design matrix
    set.seed(10)
    X <- cbind(1, rep(0:1, each = n / 2))
    cov_dat <- data.frame(cov = X[, 2])
    # cluster membership
    cluster <- rep(1:4, each = n/4)
    cluster_named <- paste("cage", cluster, sep = "")
    cov_dat$cluster <- cluster
    # intercepts for each category
    b0 <- rnorm(J)
    # coefficients for X1 for each category
    b1 <- seq(1, 5, length.out = J)
    # mean center the coefficients
    b1 <- b1 - mean(b1)
    # set the coefficient for the 3rd category to 4 (why not!?)
    # Note that because of the constraint, we're only able to estimate
    # b1 - mean(b1), which is ~3.9.
    b1[3] <- 4
    # generate B coefficient matrix
    b <- rbind(b0, b1)

    # simulate data according to a zero-inflated negative binomial distribution
    # the mean model used to simulate this data takes into account the cluster membership
    Y <- radEmu:::simulate_data(n = n,
                                J = J,
                                b0 = b0,
                                b1 = b1,
                                distn = "ZINB",
                                zinb_size = 10,
                                zinb_zero_prop = 0.3,
                                mean_z = 5,
                                X = X,
                                cluster = cluster)

Let’s just pause to look at the elements of `cluster`:

    table(cluster_named)
    #> cluster_named
    #> cage1 cage2 cage3 cage4
    #>    15    15    15    15

So all of the observations from the first cage (or person, tank, whatever…) have `cage1` in their corresponding `cluster_named` variable.

Let’s fit a model to this data! We know that the log-fold difference between in the abundance of category 3 when comparing samples that differ by 1 unit in \(X\) is 3.5, so if we have good power, we will reject the null that \(\beta_{X_1, 3} = 0\). We fit a model including cluster dependence as follows:

    ef_cluster <- emuFit(formula = ~ cov,
                         data = cov_dat,
                         Y = Y,
                         cluster = cluster_named,
                         test_kj = data.frame(k = 2, j = 3),
                         match_row_names = FALSE)

    ef_cluster$coef[3, ]
    #>   covariate   category category_num estimate        se   lower    upper
    #> 3       cov category_3            3 4.534209 0.4675388 3.61785 5.450568
    #>   score_stat      pval
    #> 3   2.874715 0.0899809

You can check out the full object, but our estimate is 4.5 and a p-value for testing that this parameter equals zero is 0.09. Not too shabby, especially considering that about half of our observations are zero, and we have a lot of noise in our data (arising from a negative binomial simulation scheme).

Let’s also compare that to a situation where we mistakenly ignore clustering. In this case, we expect to have a smaller p-value, because we are saying that we have more independent observations.

    ef_no_cluster <- emuFit(formula = ~ cov,
                            data = cov_dat,
                            Y = Y,
                            test_kj = data.frame(k = 2, j = 3),
                            match_row_names = FALSE)
    ef_no_cluster$coef[3, ]
    #>   covariate   category category_num estimate        se    lower    upper
    #> 3       cov category_3            3 4.534209 0.3235296 3.900103 5.168316
    #>   score_stat         pval
    #> 3   17.29419 3.201648e-05

When we ignore clustering, we get an estimate of the log fold difference in category 3 across values of our covariate of 4.5 and a p-value of 0. So we can see that our estimates are the same whether or not we account for cluster, but our p-values are different (because we are pretending that we have more evidence in the absence of clustering).