# Introduction to radEmu with phyloseq

#### David Clausen, 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

This vignette provides an introduction to using `radEmu` for differential abundance analysis using a `phyloseq` data object. For more in-depth explanations of how this software works, an example of running hypothesis tests, and details on this analysis, see the vignette “intro_radEmu.Rmd”.

In this lab we’ll explore a dataset published by Wirbel et al. (2019). This is a meta-analysis of case-control studies, meaning that Wirbel et al. collected raw sequencing data from studies other researchers conducted and re-analyzed it (in this case, they also collected some new data of their own).

Wirbel et al. published two pieces of data we’ll focus on today:

- metadata giving demographics and other information about participants
- a mOTU (metagenomic OTU) table

In the manuscript, we looked at differential abundance across otherwise similar colorectal cancer and non-cancer control study participants for the 849 mOTUs that Wirbel et al. published. For the purpose of having a streamlined tutorial, we will only look at a subset of those 849 mOTUs in this vignette.

## Loading and exploring data

Note that in order to follow along with this tutorial (but not to use `radEmu`!) you will need to have `phyloseq` installed. We will check if you have `phyloseq` installed, and if you do not then you can read the following code but it will not be run.

    phy <- requireNamespace("phyloseq", quietly = TRUE) == TRUE

    #> [1] "phyloseq is installed: TRUE"

Now that we have loaded the `phyloseq` package, we will create our `phyloseq` data object.

    data(wirbel_sample)
    data(wirbel_otu)
    data(wirbel_taxonomy)
    wirbel_phylo <- phyloseq::phyloseq(phyloseq::sample_data(wirbel_sample),
                                       phyloseq::otu_table(wirbel_otu, taxa_are_rows = FALSE),
                                       phyloseq::tax_table(wirbel_taxonomy))
    wirbel_phylo
    #> phyloseq-class experiment-level object
    #> otu_table()   OTU Table:         [ 845 taxa and 566 samples ]
    #> sample_data() Sample Data:       [ 566 samples by 14 sample variables ]
    #> tax_table()   Taxonomy Table:    [ 845 taxa by 7 taxonomic ranks ]

We’ll start by looking at the metadata.

    dim(phyloseq::sample_data(wirbel_phylo))
    #> [1] 566  14
    head(phyloseq::sample_data(wirbel_phylo))
    #>                             Sample_ID External_ID Age Gender BMI Country  Study
    #> CCIS00146684ST.4.0 CCIS00146684ST-4-0      FR-726  72      F  25     FRA FR-CRC
    #> CCIS00281083ST.3.0 CCIS00281083ST-3-0      FR-060  53      M  32     FRA FR-CRC
    #> CCIS02124300ST.4.0 CCIS02124300ST-4-0      FR-568  35      M  23     FRA FR-CRC
    #> CCIS02379307ST.4.0 CCIS02379307ST-4-0      FR-828  67      M  28     FRA FR-CRC
    #> CCIS02856720ST.4.0 CCIS02856720ST-4-0      FR-027  74      M  27     FRA FR-CRC
    #> CCIS03473770ST.4.0 CCIS03473770ST-4-0      FR-192  29      M  24     FRA FR-CRC
    #>                    Group Library_Size Age_spline.1 Age_spline.2 BMI_spline.1
    #> CCIS00146684ST.4.0   CTR     35443944  -0.19755428    0.7389621   1.18982420
    #> CCIS00281083ST.3.0   CTR     19307896  -0.08126128   -0.6818534  -1.40679307
    #> CCIS02124300ST.4.0   CTR     42141246  -2.17453529   -0.6818534   0.45476676
    #> CCIS02379307ST.4.0   CRC      4829533   0.67464323   -0.1490476   0.07698823
    #> CCIS02856720ST.4.0   CTR     34294675  -0.54643328    1.0941660   0.44793355
    #> CCIS03473770ST.4.0   CTR     20262319  -2.87229329   -0.6818534   0.95261443
    #>                    BMI_spline.2 Sampling
    #> CCIS00146684ST.4.0   -0.5606919   BEFORE
    #> CCIS00281083ST.3.0    2.0039136   BEFORE
    #> CCIS02124300ST.4.0   -0.6706035   BEFORE
    #> CCIS02379307ST.4.0    0.5384247   BEFORE
    #> CCIS02856720ST.4.0    0.1720525   BEFORE
    #> CCIS03473770ST.4.0   -0.6706035   BEFORE

We can see that this dataset includes \(566\) observations and \(14\) variables.

Now let’s load the mOTU table.

    dim(phyloseq::otu_table(wirbel_phylo))
    #> [1] 566 845
    # let's check out a subset
    phyloseq::otu_table(wirbel_phylo)[1:5, 1:3]
    #> OTU Table:          [3 taxa and 5 samples]
    #>                      taxa are columns
    #>                    Streptococcus anginosus [ref_mOTU_v2_0004]
    #> CCIS00146684ST.4.0                                          0
    #> CCIS00281083ST.3.0                                          0
    #> CCIS02124300ST.4.0                                          2
    #> CCIS02379307ST.4.0                                          0
    #> CCIS02856720ST.4.0                                          1
    #>                    Enterobacteriaceae sp. [ref_mOTU_v2_0036]
    #> CCIS00146684ST.4.0                                         3
    #> CCIS00281083ST.3.0                                         0
    #> CCIS02124300ST.4.0                                         5
    #> CCIS02379307ST.4.0                                         5
    #> CCIS02856720ST.4.0                                       675
    #>                    Citrobacter sp. [ref_mOTU_v2_0076]
    #> CCIS00146684ST.4.0                                  0
    #> CCIS00281083ST.3.0                                  0
    #> CCIS02124300ST.4.0                                  0
    #> CCIS02379307ST.4.0                                  0
    #> CCIS02856720ST.4.0                                  0

We can see that this table has \(566\) samples (just like the metadata) and \(845\) mOTUs. Let’s save these mOTU names in a vector.

    mOTU_names <- colnames(phyloseq::otu_table(wirbel_phylo))

Finally, we can check out the taxonomy table.

    head(phyloseq::tax_table(wirbel_phylo))
    #> Taxonomy Table:     [6 taxa by 7 taxonomic ranks]:
    #>                                                     domain     phylum
    #> Streptococcus anginosus [ref_mOTU_v2_0004]          "Bacteria" "Bacillota"
    #> Enterobacteriaceae sp. [ref_mOTU_v2_0036]           "Bacteria" "Pseudomonadota"
    #> Citrobacter sp. [ref_mOTU_v2_0076]                  "Bacteria" "Pseudomonadota"
    #> Klebsiella michiganensis/oxytoca [ref_mOTU_v2_0079] "Bacteria" "Pseudomonadota"
    #> Enterococcus faecalis [ref_mOTU_v2_0116]            "Bacteria" "Bacillota"
    #> Lactobacillus salivarius [ref_mOTU_v2_0125]         "Bacteria" "Bacillota"
    #>                                                     class
    #> Streptococcus anginosus [ref_mOTU_v2_0004]          "Bacilli"
    #> Enterobacteriaceae sp. [ref_mOTU_v2_0036]           "Gammaproteobacteria"
    #> Citrobacter sp. [ref_mOTU_v2_0076]                  "Gammaproteobacteria"
    #> Klebsiella michiganensis/oxytoca [ref_mOTU_v2_0079] "Gammaproteobacteria"
    #> Enterococcus faecalis [ref_mOTU_v2_0116]            "Bacilli"
    #> Lactobacillus salivarius [ref_mOTU_v2_0125]         "Bacilli"
    #>                                                     order
    #> Streptococcus anginosus [ref_mOTU_v2_0004]          "Lactobacillales"
    #> Enterobacteriaceae sp. [ref_mOTU_v2_0036]           "Enterobacterales"
    #> Citrobacter sp. [ref_mOTU_v2_0076]                  "Enterobacterales"
    #> Klebsiella michiganensis/oxytoca [ref_mOTU_v2_0079] "Enterobacterales"
    #> Enterococcus faecalis [ref_mOTU_v2_0116]            "Lactobacillales"
    #> Lactobacillus salivarius [ref_mOTU_v2_0125]         "Lactobacillales"
    #>                                                     family
    #> Streptococcus anginosus [ref_mOTU_v2_0004]          "Streptococcaceae"
    #> Enterobacteriaceae sp. [ref_mOTU_v2_0036]           "Enterobacteriaceae"
    #> Citrobacter sp. [ref_mOTU_v2_0076]                  "Enterobacteriaceae"
    #> Klebsiella michiganensis/oxytoca [ref_mOTU_v2_0079] "Enterobacteriaceae"
    #> Enterococcus faecalis [ref_mOTU_v2_0116]            "Enterococcaceae"
    #> Lactobacillus salivarius [ref_mOTU_v2_0125]         "Lactobacillaceae"
    #>                                                     genus
    #> Streptococcus anginosus [ref_mOTU_v2_0004]          "Streptococcus"
    #> Enterobacteriaceae sp. [ref_mOTU_v2_0036]           "Enterobacteriaceae"
    #> Citrobacter sp. [ref_mOTU_v2_0076]                  "Citrobacter"
    #> Klebsiella michiganensis/oxytoca [ref_mOTU_v2_0079] "Klebsiella"
    #> Enterococcus faecalis [ref_mOTU_v2_0116]            "Enterococcus"
    #> Lactobacillus salivarius [ref_mOTU_v2_0125]         "Lactobacillus"
    #>                                                     species
    #> Streptococcus anginosus [ref_mOTU_v2_0004]          "Streptococcus anginosus"
    #> Enterobacteriaceae sp. [ref_mOTU_v2_0036]           "Enterobacteriaceae bacterium"
    #> Citrobacter sp. [ref_mOTU_v2_0076]                  "Citrobacter sp."
    #> Klebsiella michiganensis/oxytoca [ref_mOTU_v2_0079] "Klebsiella michiganensis"
    #> Enterococcus faecalis [ref_mOTU_v2_0116]            "Enterococcus faecalis"
    #> Lactobacillus salivarius [ref_mOTU_v2_0125]         "Ligilactobacillus salivarius"

## Fitting a model

`radEmu` is a package that can be used to estimate fold-differences in the abundance of microbial taxa between levels of a covariate. In this analysis, the covariate that we are primarily interested in is whether a sample is from a case of colorectal cancer or a control. We will make control (“CTR”) the reference category:

    phyloseq::sample_data(wirbel_phylo)$Group <- factor(phyloseq::sample_data(wirbel_phylo)$Group, levels = c("CTR","CRC"))

While in general we would fit a model to all mOTUs, we are going to subset to some specific genera for the purposes of this tutorial. Let’s look at Eubacterium, Porphyromonas, Faecalibacteria, and Fusobacterium for now.

    chosen_genera <- c("Eubacterium", "Faecalibacterium", "Fusobacterium", "Porphyromonas")
    wirbel_restrict <- phyloseq::subset_taxa(wirbel_phylo, genus %in% chosen_genera)

Again, while we would generally fit a model using all of our samples, for this tutorial we are only going to consider data from a case-control study from China.

    wirbel_china <- phyloseq::subset_samples(wirbel_restrict, Country == "CHI")

Next, we want to confirm that all samples have at least one non-zero count across the categories we’ve chosen and that all categories have at least one non-zero count across the samples we’ve chosen.

    sum(rowSums(phyloseq::otu_table(wirbel_china)) == 0) # no samples have a count sum of 0
    #> [1] 0
    sum(colSums(phyloseq::otu_table(wirbel_china)) == 0) # one category has a count sum of 0
    #> [1] 1
    category_to_rm <- names(which(colSums(phyloseq::otu_table(wirbel_china)) == 0))
    wirbel_china <- phyloseq::subset_taxa(wirbel_china, species != category_to_rm)
    sum(colSums(phyloseq::otu_table(wirbel_china)) == 0) # now no categories have a count sum of 0
    #> [1] 0

The function that we use to fit our model is called `emuFit`. It can accept your data in various forms, and here we will show how to use it with a `phyloseq` object as input.

    ch_fit <- emuFit(formula = ~ Group,
                     Y = wirbel_china,
                     run_score_tests = FALSE)

The way to access estimated coefficients and confidence intervals from the model is with `ch_fit$coef`.

Now, we can easily visualize our results using the `plot.emuFit` function!

    plot(ch_fit)$plots
    #> $p1

If you’d like to see more explanations of the `radEmu` software and additional analyses of this data, check out the vignette “intro_radEmu.Rmd”.