MaAsLin 3 is the next generation of MaAsLin (Microbiome Multivariable Associations with Linear Models). This comprehensive R package efficiently determines multivariable associations between clinical metadata and microbial meta-omics features. Relative to MaAsLin 2, MaAsLin 3 introduces the ability to quantify and test for both abundance and prevalence associations while accounting for compositionality. By incorporating generalized linear models, MaAsLin 3 accommodates most modern epidemiological study designs including cross-sectional and longitudinal studies.
If you use the MaAsLin 3 software, please cite our manuscript:
William A. Nickols, Thomas Kuntz, Jiaxian Shen, Sagun Maharjan, Himel Mallick, Eric A. Franzosa, Kelsey N. Thompson, Jacob T. Nearing, Curtis Huttenhower. MaAsLin 3: Refining and extending generalized multivariable linear models for meta-omic association discovery. bioRxiv 2024.12.13.628459; doi: https://doi.org/10.1101/2024.12.13.628459
The user manual can be found here. If using vignettes, users should start with the maaslin3_tutorial.Rmd vignette and then refer to the maaslin3_manual.Rmd vignette as necessary. If you have questions, please direct them to the MaAsLin Forum
R is a programming language specializing in statistical computing and graphics. You can use R just the same as any other programming languages, but it is most useful for statistical analyses, with well-established packages for common tasks such as linear modeling, ’omic data analysis, machine learning, and visualization.
You can download and install the free R software environment here. Note that you should download the latest release - this will ensure the R version you have is compatible with MaAsLin 3.
RStudio is a freely available IDE (integrated development environment) for R. It is a “wrapper” around R with some additional functionalities that make programming in R a bit easier. Feel free to download RStudio and explore its interface - but it is not required for this tutorial.
If you already have R installed, then it is possible that the R version you have does not support MaAsLin 3. The easiest way to check this is to launch R/RStudio, and in the console (“Console” pane in RStudio), type in the following command (without the > symbol):
sessionInfo()The first line of output message should indicate your current R version. For example:
> sessionInfo()
R version 4.4.1 (2024-06-14)For MaAsLin 3 to install, you will need R >= 4.4. If your version is older than that, please refer to section Installing R for the first time to download the latest R. Note that RStudio users will need to have RStudio “switch” to this later version once it is installed. This should happen automatically for Windows and Mac OS users when you relaunch RStudio. For Linux users you might need to bind the correct R executable. Either way, once you have the correct version installed, launch the software and use sessionInfo() to make sure that you indeed have R >= 4.4.
The latest version of MaAsLin 3 can be installed from BiocManager.
if (!require("BiocManager", quietly = TRUE))
install.packages("BiocManager")
BiocManager::install("biobakery/maaslin3")To compile vignettes yourself, specify dependencies = TRUE.
To run MaAsLin 3, the user must supply a table of per-sample feature abundances (with zeros still included), a table of per-sample metadata, and a model specifying how the metadata should relate to the feature prevalence (how likely the feature is to be present or absent) and abundance (how much of the feature is there if it’s there). MaAsLin 3 will return a table of associations including an effect size and p-value for each feature-metadatum association and a folder of visuals including a summary plot and diagnostic plots for significant associations.
MaAsLin3 requires two input files.
The data file can contain samples not included in the metadata file (along with the reverse case). For both cases, those samples not included in both files will be removed from the analysis. Also, the samples do not need to be in the same order in the two files.
Samples with NA values for the metadata will be excluded when fitting the models. It is recommended to drop these samples in advance under a missing-at-random assumption or use multiple imputation to run the complete dataset.
Example input files can be found in the inst/extdata folder of the MaAsLin 3 source. The files provided were generated from the Human Microbiome Project 2 (HMP2) data which can be downloaded from https://ibdmdb.org/.
HMP2_taxonomy.tsv: a tab-delimited file with samples as rows and species as columns. It is a subset of the full HMP2 taxonomy that includes just some of the the species abundances.HMP2_metadata.tsv: a tab-delimited file with samples as rows and metadata as columns. It is a subset of the full HMP2 metadata that includes just some of the fields.# Read abundance table
taxa_table_name <- system.file("extdata", "HMP2_taxonomy.tsv",
package = "maaslin3")
taxa_table <- read.csv(taxa_table_name, sep = '\t')
# Read metadata table
metadata_name <- system.file("extdata", "HMP2_metadata.tsv",
package = "maaslin3")
metadata <- read.csv(metadata_name, sep = '\t')
# Factor the categorical variables to test IBD against healthy controls
metadata$diagnosis <-
factor(metadata$diagnosis, levels = c('nonIBD', 'UC', 'CD'))
metadata$dysbiosis_state <-
factor(metadata$dysbiosis_state, levels =
c('none', 'dysbiosis_UC', 'dysbiosis_CD'))
metadata$antibiotics <-
factor(metadata$antibiotics, levels = c('No', 'Yes'))
taxa_table[1:5, 1:5]## Phocaeicola_vulgatus Faecalibacterium_prausnitzii
## CSM5FZ3N_P 0.4265226 0.060255109
## CSM5FZ3R_P 0.5369584 0.007396904
## CSM5FZ3T_P 0.5911821 0.000000000
## CSM5FZ3V_P 0.2661378 0.029680329
## CSM5FZ3X_P 0.6601039 0.003596740
## Bacteroides_uniformis Prevotella_copri_clade_A Bacteroides_stercoris
## CSM5FZ3N_P 0.2692411314 0.000000e+00 0.000000000
## CSM5FZ3R_P 0.2526048469 0.000000e+00 0.008390958
## CSM5FZ3T_P 0.0000000000 0.000000e+00 0.000000000
## CSM5FZ3V_P 0.4004265260 0.000000e+00 0.000000000
## CSM5FZ3X_P 0.0008804283 1.308102e-05 0.001335669metadata[1:5, 1:5]## participant_id site_name week_num reads diagnosis
## CSM5FZ3N_P C3001 Cedars-Sinai 0 9961743 CD
## CSM5FZ3R_P C3001 Cedars-Sinai 2 16456391 CD
## CSM5FZ3T_P C3002 Cedars-Sinai 0 10511448 CD
## CSM5FZ3V_P C3001 Cedars-Sinai 6 17808965 CD
## CSM5FZ3X_P C3002 Cedars-Sinai 2 13160893 CDMaAsLin 3 is run by specifying the abundance table (input_data), the metadata table (input_metadata), the output directory (output), and a model. The model can come from a formula or vectors of terms. In either case, variable names should not have spaces or unusual characters.
formula parameter should be set to any formula that satisfies the lme4 specifications: fixed effects, random effects (section 4.2), interaction terms (section 4.3), polynomial terms, and more can all be included. If categorical variables are included as fixed effects, each level will be tested against the first factor level. In addition, ordered predictors (section 4.4) and group predictors (section 4.5) can be included by including group(variable_name) and ordered(variable_name) in the formula. When the data involve paired or matched case-control samples, conditional logistic regression can be run by specifying the grouping variable with strata(variable_name).fixed_effects, random_effects, group_effects, ordered_effects, and strata_effects. Categorical variables should either be ordered as factors beforehand, or reference should be provided as a string of ‘variable,reference’ semi-colon delimited for multiple variables (e.g., variable_1,reference_1;variable_2,reference_2). NOTE: adding a space between the variable and level might result in the wrong reference level being used.Because MaAsLin 3 identifies prevalence (presence/absence) associations, sample read depth (number of reads) should be included as a covariate if available. Deeper sequencing will likely increase feature detection in a way that could spuriously correlate with metadata of interest when read depth is not included in the model.
The following command runs MaAsLin 3 on the HMP2 data, running a multivariable regression model to test for the association between microbial species abundance and prevalence versus IBD diagnosis and dysbiosis scores while controlling for antibiotic usage, age, and the sampling depth (reads). The abundance associations come from fitting multivariate linear models to the log base 2 relative abundances of features in samples in which the feature is present. The prevalence associations come from fitting multivariate logistic models to the presence/absence data for a feature across all samples. Outputs are directed to a folder called hmp2_output under the current working directory (output = "hmp2_output").
To show one of each type of plot (see below), the maximum number of significant associations to plot has been increased with max_pngs=100 (default 30). All other parameters beyond the first four are the default parameters but are still included for clarity:
normalization = 'TSS') with a log transformation ('transform = 'LOG', base 2) afterwards will be used. These are almost always the recommended choices (and all MaAsLin 3 evaluations were performed with these options), but other normalizations and transformations are allowed (see normalization and transform in ?maaslin3).augment = TRUE). This is almost always recommended, though it can be turned off (see augment in the manual).standardize = TRUE so that the coefficients will be on the same scale (improving visualization).max_significance = 0.1) determines which associations are significant, but this can also be changed (see max_significance in the manual).median_comparison_abundance). Since prevalence coefficients do not have the same compositional properties, they are instead compared against 0 (median_comparison_prevalence). See below for a discussion of these parameters.cores = 1).The abundance, prevalence, and variance filtering parameters are not included, and they are 0 by default. Different from other differential abundance tools, low prevalence features need not be filtered out since the prevalence modeling in MaAsLin 3 already accounts for high proportions of zeros. However, filtering low prevalence features might improve power.
set.seed(1)
fit_out <- maaslin3(input_data = taxa_table,
input_metadata = metadata,
output = 'hmp2_output',
formula = '~ diagnosis + dysbiosis_state +
antibiotics + age + reads',
normalization = 'TSS',
transform = 'LOG',
augment = TRUE,
standardize = TRUE,
max_significance = 0.1,
median_comparison_abundance = TRUE,
median_comparison_prevalence = FALSE,
max_pngs = 10,
cores = 1)By default, many lines of information are printed while the models fit, but the verbosity can be changed with the verbosity argument (e.g., verbosity = 'WARN').
MaAsLin 3 can also take as input a SummarizedExperiment or TreeSummarizedExperiment object from BioConductor:
se <- SummarizedExperiment(
assays = list(taxa_table = t(taxa_table)),
colData = metadata
)
fit_out <- maaslin3(input_data = se,
output = 'hmp2_output',
formula = '~ diagnosis + dysbiosis_state +
antibiotics + age + reads',
normalization = 'TSS',
transform = 'LOG',
augment = TRUE,
standardize = TRUE,
max_significance = 0.1,
median_comparison_abundance = TRUE,
median_comparison_prevalence = FALSE,
max_pngs = 10,
cores = 1)When median_comparison_abundance or median_comparison_prevalence are on, the coefficients for a metadatum will be tested against the median coefficient for that metadatum (median across the features). Otherwise, the coefficients will be tested against 0. For abundance associations, this is designed to account for compositionality, the idea that testing against zero on the relative scale is not equivalent to testing against zero on the absolute scale. The user manual contains a more extensive discussion of when to use median_comparison_abundance and median_comparison_prevalence, but in summary:
median_comparison_abundance is TRUE by default and should be used to make inference on the absolute scale when using relative abundance data. When median_comparison_abundance is TRUE, only the p-values and q-values change; the coefficients returned are still the relative abundance coefficients unless subtract_median is TRUE in which case the median will be subtracted from the coefficients.median_comparison_abundance should be FALSE when (1) testing for significant relative associations, (2) testing for absolute abundance associations under the assumption that the total absolute abundance is not changing, or (3) testing for significant absolute associations when supplying spike-in or total abundances with unscaled_abundance.median_comparison_prevalence is FALSE by default.The main output from MaAsLin 3 is the list of significant associations in significant_results.tsv. This lists all associations with joint or individual q-values that pass MaAsLin 3’s significance threshold, ordered by increasing individual q-value. The format is:
feature and metadata are the feature and metadata names.value and name are the value of the metadata and variable name from the model.coef and stderr are the fit coefficient and standard error from the model. In abundance models, a one-unit change in the metadatum variable corresponds to a \(2^{\textrm{coef}}\) fold change in the relative abundance of the feature. In prevalence models, a one-unit change in the metadatum variable corresponds to a \(\textrm{coef}\) change in the log-odds of a feature being present.pval_individual and qval_individual are the p-value and false discovery rate (FDR) corrected q-value of the individual association. FDR correction is performed over all p-values without errors in the abundance and prevalence modeling together.pval_joint and qval_joint are the p-value and q-value of the joint prevalence and abundance association. The p-value comes from plugging in the minimum of the association’s abundance and prevalence p-values into the Beta(1,2) CDF. It is interpreted as the probability that either the abundance or prevalence association would be as extreme as observed if there was neither an abundance nor prevalence association between the feature and metadatum.model specifies whether the association is abundance or prevalence.N and N_not_zero are the total number of data points and the total number of non-zero data points for the feature.| feature | metadata | value | name | coef | stderr | pval_individual | qval_individual | pval_joint | qval_joint | model | N | N_not_zero |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Phocaeicola_sartorii | reads | reads | reads | 1.1 | 0.0787 | 5.42e-44 | 1.06e-40 | 1.08e-43 | 1.14e-40 | prevalence | 1530 | 424 |
| Faecalibacterium_prausnitzii | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | -3.49 | 0.282 | 3.54e-35 | 3.46e-32 | 7.09e-35 | 3.74e-32 | prevalence | 1530 | 1370 |
| Bifidobacterium_longum | age | age | age | -0.672 | 0.0594 | 1.01e-29 | 4.94e-27 | 2.02e-29 | 7.12e-27 | prevalence | 1530 | 843 |
| Clostridium_sp_AM49_4BH | diagnosis | CD | diagnosisCD | -1.81 | 0.163 | 1.11e-28 | 4.34e-26 | 2.22e-28 | 5.87e-26 | prevalence | 1530 | 344 |
| Phocaeicola_dorei | reads | reads | reads | 0.66 | 0.0634 | 2.56e-25 | 8.35e-23 | 5.13e-25 | 1.08e-22 | prevalence | 1530 | 769 |
| GGB16040_SGB9347 | age | age | age | 0.96 | 0.0944 | 2.65e-24 | 7.39e-22 | 5.29e-24 | 9.31e-22 | prevalence | 1530 | 114 |
| Firmicutes_bacterium_AF16_15 | diagnosis | UC | diagnosisUC | -1.67 | 0.166 | 6.77e-24 | 1.65e-21 | 1.35e-23 | 2.04e-21 | prevalence | 1530 | 827 |
| Eubacterium_rectale | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | -2.36 | 0.237 | 2.47e-23 | 5.36e-21 | 4.94e-23 | 6.51e-21 | prevalence | 1530 | 1210 |
| Phascolarctobacterium_faecium | age | age | age | 0.574 | 0.0578 | 3.19e-23 | 6.24e-21 | 6.39e-23 | 7.5e-21 | prevalence | 1530 | 503 |
| Dysosmobacter_welbionis | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | -2.4 | 0.242 | 3.69e-23 | 6.56e-21 | 7.38e-23 | 7.8e-21 | prevalence | 1530 | 1190 |
| Blautia_faecis | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | -2.42 | 0.249 | 2.66e-22 | 4.33e-20 | 5.31e-22 | 5.1e-20 | prevalence | 1530 | 1120 |
| Fusicatenibacter_saccharivorans | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | -2.35 | 0.243 | 4.52e-22 | 6.79e-20 | 9.03e-22 | 7.95e-20 | prevalence | 1530 | 1120 |
| Lachnospira_sp_NSJ_43 | diagnosis | CD | diagnosisCD | -1.65 | 0.177 | 8.94e-21 | 1.16e-18 | 1.79e-20 | 1.45e-18 | prevalence | 1530 | 296 |
| Bacteroides_ovatus | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | -2.13 | 0.228 | 1.09e-20 | 1.33e-18 | 2.18e-20 | 1.65e-18 | prevalence | 1530 | 1180 |
| Clostridium_sp_AM49_4BH | diagnosis | UC | diagnosisUC | -1.57 | 0.17 | 2.51e-20 | 2.89e-18 | 5.02e-20 | 3.54e-18 | prevalence | 1530 | 344 |
| Bacteroides_faecis | reads | reads | reads | 0.6 | 0.0657 | 7.46e-20 | 8.1e-18 | 1.49e-19 | 9.84e-18 | prevalence | 1530 | 434 |
| Dysosmobacter_welbionis | reads | reads | reads | 0.719 | 0.0798 | 2.1e-19 | 2.05e-17 | 4.2e-19 | 2.61e-17 | prevalence | 1530 | 1190 |
| Roseburia_sp_AF02_12 | diagnosis | CD | diagnosisCD | -1.38 | 0.154 | 2.44e-19 | 2.27e-17 | 4.87e-19 | 2.86e-17 | prevalence | 1530 | 363 |
| Clostridiales_bacterium | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | -2.46 | 0.275 | 3.56e-19 | 3.16e-17 | 7.12e-19 | 3.96e-17 | prevalence | 1530 | 985 |
| Alistipes_putredinis | diagnosis | UC | diagnosisUC | -1.42 | 0.159 | 5.06e-19 | 4.3e-17 | 1.01e-18 | 5.35e-17 | prevalence | 1530 | 873 |
MaAsLin 3 generates two types of output files explained below: data and visualizations. In addition, the object returned from maaslin3 (fit_out above) contains all the data and results (see ?maaslin_fit).
significant_results.tsv
all_results.tsv
features
models_linear.rds and models_logistic.rds
linear for linear models, logistic for logistic models).save_models is set to TRUE.residuals_linear.rds and residuals_logstic.rds
fitted_linear.rds and fitted_logistic.rds
ranef_linear.rds and ranef_logistic.rds
maaslin3.log
summary_plot.pdf
max_significance, and two stars indicate the individual q-value is below max_significance divided by 10. (Make sure to set include=T to view the plots when knitting the Rmd.)association_plots/[metadatum]/[association]/ [metadatum]_[feature]_[association].png
max_pngs.max_pngs = 250 above and set eval=TRUE and include=TRUE in this chunkAt the top right of each association plot is the name of the significant association in the results file, the FDR corrected q-value for the individual association, the number of samples in the dataset, and the number of samples with non-zero abundances for the feature. In the plots with categorical metadata variables, the reference category is on the left, and the significant q-values and coefficients in the top right are in the order of the values specified above. Because the displayed coefficients correspond to the full fit model with (possibly) scaled metadata variables, the marginal association plotted might not match the coefficient displayed. However, the plots are intended to provide an interpretable visual while usually agreeing with the full model.
There are a few common issues to check for in the results:
error column of all_results.tsv. Often, these indicate model fitting failures or poor fits that should not be trusted, but sometimes the warnings can be benign, and the model fit might still be reasonable. Users should check associations of interest if they produce errors.
warn_prevalence=TRUE, a note will be produced in the error column indicating when prevalence associations are likely induced by abundance associations. These should be checked visually with the diagnosic visuals.association_plots directory.
Once maaslin3 has been run once, maaslin_plot_results_from_output can be run to (re-)create the plots. This allows the user to plot the associations even without having the R object returned by maaslin_fit or maaslin3 (e.g., if fitting models through the command line). It is recommended to fit models with simple variables names that are robust to formula plotting and then convert these into proper names for plotting. Likewise, heatmap_vars and coef_plot_vars can be specified at any point, but it is usually easier to see how the names come out first and then choose which metadata variables will go in the coefficient plot and which will go in the heatmap afterwards with maaslin_plot_results_from_output.
# This section is necessary for updating the
# summary plot and the association plots
# Rename results file with clean titles
all_results <- read.csv('hmp2_output/all_results.tsv', sep='\t')
all_results <- all_results %>%
mutate(metadata = case_when(metadata == 'age' ~ 'Age',
metadata == 'antibiotics' ~ 'Abx',
metadata == 'diagnosis' ~ 'Diagnosis',
metadata == 'dysbiosis_state' ~ 'Dysbiosis',
metadata == 'reads' ~ 'Read depth'),
value = case_when(value == 'dysbiosis_CD' ~ 'CD',
value == 'dysbiosis_UC' ~ 'UC',
value == 'Yes' ~ 'Used', # Antibiotics
value == 'age' ~ 'Age',
value == 'reads' ~ 'Read depth',
TRUE ~ value),
feature = gsub('_', ' ', feature) %>%
gsub(pattern = 'sp ', replacement = 'sp. '))
# Write results
write.table(all_results, 'hmp2_output/all_results.tsv', sep='\t')
# Set the new heatmap and coefficient plot variables and order them
heatmap_vars = c('Dysbiosis UC', 'Diagnosis UC',
'Abx Used', 'Age', 'Read depth')
coef_plot_vars = c('Dysbiosis CD', 'Diagnosis CD')
# This section is necessary for updating the association plots
taxa_table_copy <- taxa_table
colnames(taxa_table_copy) <- gsub('_', ' ', colnames(taxa_table_copy)) %>%
gsub(pattern = 'sp ', replacement = 'sp. ')
# Rename the features in the norm transformed data file
data_transformed <-
read.csv('hmp2_output/features/data_transformed.tsv', sep='\t')
colnames(data_transformed) <-
gsub('_', ' ', colnames(data_transformed)) %>%
gsub(pattern = 'sp ', replacement = 'sp. ')
write.table(data_transformed,
'hmp2_output/features/data_transformed.tsv',
sep='\t', row.names = FALSE)
# Rename the metadata like in the outputs table
metadata_copy <- metadata
colnames(metadata_copy) <-
case_when(colnames(metadata_copy) == 'age' ~ 'Age',
colnames(metadata_copy) == 'antibiotics' ~ 'Abx',
colnames(metadata_copy) == 'diagnosis' ~ 'Diagnosis',
colnames(metadata_copy) == 'dysbiosis_state' ~ 'Dysbiosis',
colnames(metadata_copy) == 'reads' ~ 'Read depth',
TRUE ~ colnames(metadata_copy))
metadata_copy <- metadata_copy %>%
mutate(Dysbiosis = case_when(Dysbiosis == 'dysbiosis_UC' ~ 'UC',
Dysbiosis == 'dysbiosis_CD' ~ 'CD',
Dysbiosis == 'none' ~ 'None') %>%
factor(levels = c('None', 'UC', 'CD')),
Abx = case_when(Abx == 'Yes' ~ 'Used',
Abx == 'No' ~ 'Not used') %>%
factor(levels = c('Not used', 'Used')),
Diagnosis = case_when(Diagnosis == 'nonIBD' ~ 'non-IBD',
TRUE ~ Diagnosis) %>%
factor(levels = c('non-IBD', 'UC', 'CD')))
# Recreate the plots
scatter_plots <- maaslin_plot_results_from_output(
output = 'hmp2_output',
metadata = metadata_copy,
normalization = "TSS",
transform = "LOG",
median_comparison_abundance = TRUE,
median_comparison_prevalence = FALSE,
max_significance = 0.1,
max_pngs = 20)In the new summary plot below, we can see that the feature names are cleaned up, the metadata names are cleaned up, the set of metadata variables used in the coefficient plot is different, and the metadata used in the heatmap is reordered.
In the association plots, the taxa and metadata have been renamed to be consistent with the results file from earlier.
Most microbiome methodologies have historically focused on relative abundances (proportions out of 1). However, some experimental protocols can enable estimation of absolute abundances (cell count/concentration). MaAsLin 3 can be used with two types of absolute abundance estimation: spike-ins and total abundance scaling. In a spike-in procedure, a small, known quantity of a microbe that otherwise would not be present in the sample is added, and the sequencing procedure is carried out as usual. Then, the absolute abundance of a microbe already in the community is estimated as: \[\textrm{Absolute abundance other microbe}=\frac{\textrm{Relative abundance other microbe}}{\textrm{Relative abundance spike-in microbe}}\cdot (\textrm{Absolute abundance spike-in microbe})\] Alternatively, the total microbial abundance of a sample can be determined (e.g., with qPCR of a marker gene or by cell counting). Then, the absolute abundance of a microbe in the community is estimated as: \[\textrm{Absolute abundance microbe}=(\textrm{Total absolute abundance})\cdot(\textrm{Relative abundance microbe})\]
The following section shows a synthetic spike-in procedure with simulated data from SparseDOSSA2. The abundance table is like any other abundance table input to MaAsLin 3 except that ‘Feature101’ is the spike-in (which must be present in every sample). The scaling factor data frame has ‘Feature101’ as its only column name with the absolute abundance of the spike-in for each sample (rownames). If the same quantity of the spike-in was added to all samples, this column could be entirely 1 (or any other arbitrary value). When using a spike-in procedure, the scaling factor data frame should have a single column named the same as one of the features in the abundance table, which will be used as the spike-in.
# Abundance table
taxa_table_name <- system.file("extdata", "abundance_spike_in_ex.tsv",
package = "maaslin3")
spike_in_taxa_table <- read.csv(taxa_table_name, sep = '\t')
# Metadata table
metadata_name <- system.file("extdata", "metadata_spike_in_ex.tsv",
package = "maaslin3")
spike_in_metadata <- read.csv(metadata_name, sep = '\t')
for (col in c('Metadata_1', 'Metadata_2', 'Metadata_5')) {
spike_in_metadata[,col] <- factor(spike_in_metadata[,col])
}
# Spike-in table
unscaled_name <- system.file("extdata",
"scaling_factors_spike_in_ex.tsv",
package = "maaslin3")
spike_in_unscaled <- read.csv(unscaled_name, sep = '\t')
spike_in_taxa_table[c(1:5, 101),1:5]## Sample1 Sample2 Sample3 Sample4 Sample5
## Feature1 0 0 0 0 0
## Feature2 0 0 0 0 0
## Feature3 554 0 1737 0 0
## Feature4 0 0 0 0 0
## Feature5 0 0 0 0 0
## Feature101 15464 4788 5155 2052 11540spike_in_metadata[1:5,]## Metadata_1 Metadata_2 Metadata_3 Metadata_4 Metadata_5 ID
## Sample1 1 1 -0.41011453 0.6352468 0 Subject1
## Sample2 0 1 -1.35105918 0.2362369 0 Subject2
## Sample3 1 1 -0.48798215 -0.5458514 0 Subject3
## Sample4 0 0 -0.07268457 -0.9903428 1 Subject4
## Sample5 1 0 0.27201198 1.1333223 1 Subject5spike_in_unscaled[1:5, , drop=FALSE]## Feature101
## Sample1 418
## Sample2 183
## Sample3 38
## Sample4 3230
## Sample5 243The following code fits the absolute abundance model. Note that the normalization must be set to TSS since the procedure involves scaling the relative abundance ratios. Also, median_comparison_abundance is now set to FALSE since we want to test the absolute coefficients against zero. The data frame of absolute abundances is included as the unscaled_abundance parameter, and the spike-in strategy will automatically be detected based on the column name.
fit_out <- maaslin3(
input_data = spike_in_taxa_table,
input_metadata = spike_in_metadata,
output = 'spike_in_demo',
formula = '~ Metadata_1 + Metadata_2 + Metadata_3 +
Metadata_4 + Metadata_5',
normalization = 'TSS',
transform = 'LOG',
median_comparison_abundance = FALSE,
unscaled_abundance = spike_in_unscaled)The absolute abundance coefficients can be inspected:
rownames(fit_out$fit_data_abundance$results) <- NULL
head(fit_out$fit_data_abundance$results, 20) %>%
dplyr::mutate_if(is.numeric, .funs =
function(x){(as.character(signif(x, 3)))}) %>%
knitr::kable() %>%
kableExtra::kable_styling("striped", position = 'center') %>%
kableExtra::scroll_box(width = "800px", height = "400px")| feature | metadata | value | name | coef | stderr | pval_individual | qval_individual | pval_joint | qval_joint | error | model | N | N_not_zero |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Feature58 | Metadata_2 | 1 | Metadata_21 | -4.9 | 0.519 | 7.63e-13 | 5.48e-10 | 7.63e-13 | 3.2e-10 | NA | linear | 100 | 58 |
| Feature88 | Metadata_5 | 1 | Metadata_51 | 7.86 | 0.819 | 2.99e-12 | 1.08e-09 | 5.99e-12 | 1.26e-09 | NA | linear | 100 | 49 |
| Feature10 | Metadata_1 | 1 | Metadata_11 | -4.19 | 0.669 | 3.96e-07 | 5.29e-05 | 3.96e-07 | 4.16e-05 | NA | linear | 100 | 40 |
| Feature97 | Metadata_4 | Metadata_4 | Metadata_4 | -2.93 | 0.475 | 6.2e-06 | 0.000446 | 1.24e-05 | 0.000521 | NA | linear | 100 | 25 |
| Feature56 | Metadata_4 | Metadata_4 | Metadata_4 | 5.93 | 1 | 1.74e-05 | 0.00104 | 1.74e-05 | 0.000665 | NA | linear | 100 | 23 |
| Feature83 | Metadata_3 | Metadata_3 | Metadata_3 | 5.45 | 1.07 | 7.55e-05 | 0.00388 | 0.000151 | 0.00423 | NA | linear | 100 | 24 |
| Feature49 | Metadata_1 | 1 | Metadata_11 | -5.45 | 1.02 | 8.22e-05 | 0.00394 | 8.22e-05 | 0.00246 | NA | linear | 100 | 21 |
| Feature49 | Metadata_5 | 1 | Metadata_51 | -5.6 | 1.23 | 0.000374 | 0.0117 | 0.000748 | 0.0131 | NA | linear | 100 | 21 |
| Feature64 | Metadata_3 | Metadata_3 | Metadata_3 | -3.23 | 0.828 | 0.000612 | 0.0169 | 0.00122 | 0.0198 | NA | linear | 100 | 32 |
| Feature84 | Metadata_1 | 1 | Metadata_11 | -4.54 | 0.919 | 0.000801 | 0.0206 | 0.0016 | 0.0232 | NA | linear | 100 | 15 |
| Feature39 | Metadata_4 | Metadata_4 | Metadata_4 | -3.66 | 0.943 | 0.000867 | 0.0215 | 8.83e-07 | 6.18e-05 | NA | linear | 100 | 27 |
| Feature67 | Metadata_1 | 1 | Metadata_11 | -2.32 | 0.661 | 0.00115 | 0.0275 | 0.0023 | 0.0321 | NA | linear | 100 | 46 |
| Feature79 | Metadata_4 | Metadata_4 | Metadata_4 | -1.01 | 0.314 | 0.0045 | 0.0924 | 0.00898 | 0.122 | NA | linear | 100 | 25 |
| Feature79 | Metadata_3 | Metadata_3 | Metadata_3 | -1.07 | 0.336 | 0.00471 | 0.0941 | 0.0094 | 0.123 | NA | linear | 100 | 25 |
| Feature37 | Metadata_4 | Metadata_4 | Metadata_4 | 1 | 0.344 | 0.00525 | 0.102 | 0.0105 | 0.133 | NA | linear | 100 | 62 |
| Feature25 | Metadata_1 | 1 | Metadata_11 | -5.25 | 0.0484 | 0.00587 | 0.109 | 0.0117 | 0.142 | NA | linear | 100 | 7 |
| Feature44 | Metadata_1 | 1 | Metadata_11 | 3.73 | 0.957 | 0.00594 | 0.109 | 0.0118 | 0.142 | NA | linear | 100 | 13 |
| Feature15 | Metadata_3 | Metadata_3 | Metadata_3 | 1.93 | 0.601 | 0.00676 | 0.119 | 0.0135 | 0.149 | NA | linear | 100 | 19 |
| Feature42 | Metadata_2 | 1 | Metadata_21 | 4.08 | 1.02 | 0.00711 | 0.119 | 0.0142 | 0.149 | NA | linear | 100 | 12 |
| Feature56 | Metadata_3 | Metadata_3 | Metadata_3 | 1.86 | 0.608 | 0.00712 | 0.119 | 0.0142 | 0.149 | NA | linear | 100 | 23 |
The following section shows a synthetic total abundance scaling procedure with simulated data from SparseDOSSA2. The abundance table is like any other abundance table input to MaAsLin 3 without any extra features. The scaling factor data frame has ‘total’ as its only column name with the total absolute abundance for each sample (rownames).
# Abundance table
taxa_table_name <- system.file("extdata", "abundance_total_ex.tsv",
package = "maaslin3")
total_scaling_taxa_table <- read.csv(taxa_table_name, sep = '\t')
# Metadata table
metadata_name <- system.file("extdata", "metadata_total_ex.tsv",
package = "maaslin3")
total_scaling_metadata <- read.csv(metadata_name, sep = '\t')
for (col in c('Metadata_1', 'Metadata_3', 'Metadata_5')) {
spike_in_metadata[,col] <- factor(spike_in_metadata[,col])
}
# Total abundance table
unscaled_name <- system.file("extdata", "scaling_factors_total_ex.tsv",
package = "maaslin3")
total_scaling_unscaled <- read.csv(unscaled_name, sep = '\t')
total_scaling_taxa_table[1:5, 1:5]## Sample1 Sample2 Sample3 Sample4 Sample5
## Feature1 11837 0 0 8 68
## Feature2 0 0 0 0 1
## Feature3 0 0 0 0 0
## Feature4 0 0 1 0 0
## Feature5 0 0 0 0 0total_scaling_metadata[1:5,]## Metadata_1 Metadata_2 Metadata_3 Metadata_4 Metadata_5 ID
## Sample1 0 1.2410844 0 0.4341439 1 Subject1
## Sample2 1 0.0176896 1 1.4894610 1 Subject2
## Sample3 1 -1.7370341 1 -0.7973592 1 Subject3
## Sample4 1 0.1500793 0 3.2816068 0 Subject4
## Sample5 0 -0.2819085 0 -0.6731906 0 Subject5total_scaling_unscaled[1:5, , drop=FALSE]## total
## Sample1 1352.276
## Sample2 1720.637
## Sample3 64194.918
## Sample4 50256.129
## Sample5 1132.367The following code fits the absolute abundance model. As before, TSS must be set to TRUE, median_comparison_abundance is set to FALSE, and the data frame of absolute abundances is included as the unscaled_abundance parameter. The total abundance scaling procedure will be detected based on the column name.
fit_out <- maaslin3(
input_data = total_scaling_taxa_table,
input_metadata = total_scaling_metadata,
output = 'total_scaling_demo',
formula = '~ Metadata_1 + Metadata_2 + Metadata_3 +
Metadata_4 + Metadata_5',
normalization = 'TSS',
transform = 'LOG',
median_comparison_abundance = FALSE,
unscaled_abundance = total_scaling_unscaled)The absolute abundance coefficients can be inspected:
rownames(fit_out$fit_data_abundance$results) <- NULL
head(fit_out$fit_data_abundance$results, n = 20) %>%
dplyr::mutate_if(is.numeric,
.funs = function(x){(as.character(signif(x, 3)))}) %>%
knitr::kable() %>%
kableExtra::kable_styling("striped", position = 'center') %>%
kableExtra::scroll_box(width = "800px", height = "400px")| feature | metadata | value | name | coef | stderr | pval_individual | qval_individual | pval_joint | qval_joint | error | model | N | N_not_zero |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Feature94 | Metadata_1 | Metadata_1 | Metadata_1 | 2.83 | 0.374 | 3.32e-11 | 2.3e-08 | 6.64e-11 | 2.76e-08 | NA | linear | 100 | 94 |
| Feature80 | Metadata_3 | Metadata_3 | Metadata_3 | 2.86 | 0.316 | 3.32e-10 | 1.15e-07 | 3.32e-10 | 6.89e-08 | NA | linear | 100 | 37 |
| Feature80 | Metadata_5 | Metadata_5 | Metadata_5 | 2.22 | 0.296 | 1.92e-08 | 4.44e-06 | 3.85e-08 | 5.32e-06 | NA | linear | 100 | 37 |
| Feature66 | Metadata_2 | Metadata_2 | Metadata_2 | 3.83 | 0.56 | 1.92e-07 | 1.9e-05 | 1.92e-07 | 1.14e-05 | NA | linear | 100 | 34 |
| Feature80 | Metadata_1 | Metadata_1 | Metadata_1 | -2.09 | 0.311 | 1.69e-07 | 1.9e-05 | 1.69e-07 | 1.14e-05 | NA | linear | 100 | 37 |
| Feature4 | Metadata_1 | Metadata_1 | Metadata_1 | 2.87 | 0.434 | 1.53e-06 | 0.000106 | 3.06e-06 | 0.000127 | NA | linear | 100 | 27 |
| Feature45 | Metadata_3 | Metadata_3 | Metadata_3 | -1.82 | 0.408 | 0.000144 | 0.00712 | 0.000288 | 0.00796 | NA | linear | 100 | 32 |
| Feature101 | Metadata_1 | Metadata_1 | Metadata_1 | 1.12 | 0.287 | 0.000178 | 0.0077 | 0.000178 | 0.00568 | NA | linear | 100 | 100 |
| Feature8 | Metadata_2 | Metadata_2 | Metadata_2 | 3.33 | 0.656 | 0.000168 | 0.0077 | 0.000337 | 0.00874 | NA | linear | 100 | 20 |
| Feature5 | Metadata_4 | Metadata_4 | Metadata_4 | 4.92 | 1.23 | 0.00205 | 0.0678 | 0.00205 | 0.0406 | NA | linear | 100 | 17 |
| Feature45 | Metadata_2 | Metadata_2 | Metadata_2 | -2.08 | 0.645 | 0.00341 | 0.102 | 0.00109 | 0.025 | NA | linear | 100 | 32 |
| Feature68 | Metadata_3 | Metadata_3 | Metadata_3 | -2.27 | 0.717 | 0.00569 | 0.146 | 0.0113 | 0.168 | NA | linear | 100 | 23 |
| Feature75 | Metadata_5 | Metadata_5 | Metadata_5 | -3.29 | 1.08 | 0.00645 | 0.16 | 0.0128 | 0.184 | NA | linear | 100 | 26 |
| Feature86 | Metadata_1 | Metadata_1 | Metadata_1 | 0.974 | 0.337 | 0.00671 | 0.16 | 0.0134 | 0.185 | NA | linear | 100 | 39 |
| Feature44 | Metadata_4 | Metadata_4 | Metadata_4 | -0.565 | 0.218 | 0.011 | 0.23 | 0.011 | 0.168 | NA | linear | 100 | 100 |
| Feature79 | Metadata_2 | Metadata_2 | Metadata_2 | 1.9 | 0.724 | 0.013 | 0.257 | 0.0258 | 0.335 | NA | linear | 100 | 40 |
| Feature33 | Metadata_4 | Metadata_4 | Metadata_4 | -0.986 | 0.391 | 0.0152 | 0.291 | 0.0302 | 0.379 | NA | linear | 100 | 53 |
| Feature75 | Metadata_3 | Metadata_3 | Metadata_3 | 2.71 | 1.09 | 0.0215 | 0.381 | 0.00702 | 0.127 | NA | linear | 100 | 26 |
| Feature91 | Metadata_2 | Metadata_2 | Metadata_2 | -1.21 | 0.511 | 0.0263 | 0.444 | 0.0518 | 0.597 | NA | linear | 100 | 29 |
| Feature20 | Metadata_2 | Metadata_2 | Metadata_2 | -5.14 | 1.67 | 0.0272 | 0.445 | 0.0537 | 0.602 | NA | linear | 100 | 11 |
When using a purely computational approach to estimate absolute abundance (e.g., Nishijima et al. Cell 2024 with the function MLP), the abundance table can be estimated and then run with the normalization = 'NONE' to avoid scaling the results.
Certain studies have a natural “grouping” of sample observations, such as by subject in longitudinal designs or by family in family designs. It is important for statistic analysis to address the non-independence between samples belonging to the same group. MaAsLin 3 provides a simple interface for this with random effects, where the user can specify the grouping variable to run a mixed effect model. This grouping variable can be provided with the random_effects parameter or specified in the model with (1|grouping_variable). This will add in a “random intercept,” a per-group adjustment to the model intercept. More complicated random effects can be specified in the formula in accordance with lme4 formula parsing. For example, HMP2 has a longitudinal design where the same subject (column participant_id) can have multiple samples. We thus use participant_id as a random effect grouping variable in the model.
# Subset to only CD cases for time; taxa are subset automatically
fit_out <- maaslin3(
input_data = taxa_table,
input_metadata = metadata[metadata$diagnosis == 'CD',],
output = 'random_effects_output',
formula = '~ dysbiosis_state + antibiotics +
age + reads + (1|participant_id)',
plot_summary_plot = FALSE,
plot_associations = FALSE)## boundary (singular) fit: see help('isSingular')
## boundary (singular) fit: see help('isSingular')
## boundary (singular) fit: see help('isSingular')
## boundary (singular) fit: see help('isSingular')
## boundary (singular) fit: see help('isSingular')
## boundary (singular) fit: see help('isSingular')
## boundary (singular) fit: see help('isSingular')
## boundary (singular) fit: see help('isSingular')
## boundary (singular) fit: see help('isSingular')| feature | metadata | value | name | coef | stderr | pval_individual | qval_individual | pval_joint | qval_joint | model | N | N_not_zero |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Phocaeicola_sartorii | reads | reads | reads | 1.56 | 0.185 | 3.11e-17 | 3.37e-14 | 6.23e-17 | 3.61e-14 | prevalence | 685 | 205 |
| Faecalibacterium_prausnitzii | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | -4.89 | 0.692 | 1.68e-12 | 9.09e-10 | 3.35e-12 | 9.73e-10 | prevalence | 685 | 573 |
| Bacteroides_eggerthii | reads | reads | reads | 1.61 | 0.246 | 5.55e-11 | 2e-08 | 1.11e-10 | 2.14e-08 | prevalence | 685 | 119 |
| Blautia_faecis | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | -2.56 | 0.4 | 1.5e-10 | 4.05e-08 | 2.99e-10 | 4.34e-08 | prevalence | 685 | 469 |
| Bacteroides_uniformis | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | -3.48 | 0.556 | 4.19e-10 | 7.57e-08 | 8.38e-10 | 8.1e-08 | prevalence | 685 | 553 |
| Dysosmobacter_welbionis | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | -2.87 | 0.458 | 3.74e-10 | 7.57e-08 | 7.49e-10 | 8.1e-08 | prevalence | 685 | 524 |
| Fusicatenibacter_saccharivorans | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | -2.49 | 0.401 | 5.13e-10 | 7.94e-08 | 1.03e-09 | 8.5e-08 | prevalence | 685 | 484 |
| Parabacteroides_distasonis | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | -3.27 | 0.533 | 8.05e-10 | 1.09e-07 | 1.61e-09 | 1.17e-07 | prevalence | 685 | 484 |
| Clostridium_fessum | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | -3.59 | 0.592 | 1.29e-09 | 1.39e-07 | 2.57e-09 | 1.49e-07 | prevalence | 685 | 391 |
| Clostridium_sp_AF34_10BH | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | -3.3 | 0.543 | 1.19e-09 | 1.39e-07 | 2.38e-09 | 1.49e-07 | prevalence | 685 | 377 |
| Eubacterium_rectale | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | -2.82 | 0.469 | 1.86e-09 | 1.83e-07 | 3.72e-09 | 1.96e-07 | prevalence | 685 | 514 |
| Phocaeicola_dorei | reads | reads | reads | 0.828 | 0.139 | 2.94e-09 | 2.65e-07 | 5.88e-09 | 2.84e-07 | prevalence | 685 | 310 |
| Clostridiales_bacterium | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | -3.88 | 0.668 | 6.33e-09 | 5.28e-07 | 1.27e-08 | 5.65e-07 | prevalence | 685 | 407 |
| Clostridium_sp_AT4 | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | 3.07 | 0.593 | 1.89e-08 | 1.47e-06 | 3.79e-08 | 1.46e-06 | abundance | 685 | 229 |
| Escherichia_coli | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | 1.96 | 0.407 | 3.73e-08 | 2.53e-06 | 3.73e-08 | 1.46e-06 | abundance | 685 | 327 |
| Anaerostipes_hadrus | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | -2.31 | 0.421 | 3.7e-08 | 2.53e-06 | 7.39e-08 | 2.68e-06 | prevalence | 685 | 468 |
| Roseburia_inulinivorans | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | -2.25 | 0.413 | 4.85e-08 | 3.09e-06 | 9.7e-08 | 3.31e-06 | prevalence | 685 | 349 |
| Bacteroides_faecis | reads | reads | reads | 1.05 | 0.194 | 5.74e-08 | 3.46e-06 | 1.15e-07 | 3.7e-06 | prevalence | 685 | 203 |
| Bacteroides_thetaiotaomicron | dysbiosis_state | dysbiosis_CD | dysbiosis_statedysbiosis_CD | -3.28 | 0.608 | 6.73e-08 | 3.84e-06 | 1.35e-07 | 4.11e-06 | prevalence | 685 | 453 |
| Ruthenibacterium_lactatiformans | reads | reads | reads | 0.62 | 0.115 | 7.66e-08 | 4.15e-06 | 1.53e-07 | 4.44e-06 | prevalence | 685 | 336 |
Note that no participant_id terms are included in the outputs; the random intercepts are just used to control for grouping. If you are interested in testing the effect of time in a longitudinal study, the time point variable should be included as a fixed effect in your MaAsLin 3 formula.
Mixed effects models take longer to fit because of their complexity, and the fitting may fail more often because of the extra terms. However, it is important to account for this non-independence, and MaAsLin 3 is still able to fit complex models with thousands of samples and thousands of features in a few hours (and increasing the CPUs used with cores can help). However, random effects are not the solution to every correlated data problem. In particular, logistic random intercept models may produce poor model fits when there are fewer than 5 observations per group. In these scenarios, per-group fixed effects or, for advanced users, conditional logistic regression (see strata_effects in the manual) should be used. For example, in a before-and-after treatment study with one sample before and one sample after, the participant IDs and a pre/post indicator should be included as fixed effects or a strata variable, but only the pre/post indicator should be retained for analysis afterwards.
One of the benefits of MaAsLin 3’s formula mode is the ability to include interaction terms. Mathematically, an interaction term corresponds to the product of two columns in the design matrix. When a continuous variable is interacted with a categorical term, the interaction term corresponds to the change in the continuous variable’s slope between the categories. For two categorical variables interacted, see below; it is better explained through an example. In the formula, interactions can be specified with the : symbol to include only the interaction term(s) or the * symbol to include both the interaction term and the non-interacted terms (i.e., a*b adds the terms a, b, and a:b).
metadata <- metadata %>%
mutate(dysbiosis_general = ifelse(dysbiosis_state != 'none',
'dysbiosis', 'none')) %>%
mutate(dysbiosis_general = factor(dysbiosis_general, levels =
c('none', 'dysbiosis')))
fit_out <- maaslin3(
input_data = taxa_table,
input_metadata = metadata,
output = 'interaction_output',
formula = '~ diagnosis + diagnosis:dysbiosis_general +
antibiotics + age + reads')full_results <- rbind(fit_out$fit_data_abundance$results,
fit_out$fit_data_prevalence$results)
full_results <- full_results %>%
dplyr::arrange(qval_joint) %>%
dplyr::filter(metadata == "diagnosis")
rownames(full_results) <- NULL
head(full_results, n = 20) %>%
dplyr::mutate_if(is.numeric,
.funs = function(x){(as.character(signif(x, 3)))}) %>%
knitr::kable() %>%
kableExtra::kable_styling("striped", position = 'center') %>%
kableExtra::scroll_box(width = "800px", height = "400px")| feature | metadata | value | name | coef | stderr | pval_individual | qval_individual | pval_joint | qval_joint | error | model | N | N_not_zero |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Faecalibacterium_prausnitzii | diagnosis | CD:dysbiosis_generaldysbiosis | diagnosisCD:dysbiosis_generaldysbiosis | -2.18 | 0.309 | 1.18e-06 | 1.03e-05 | 7.2e-35 | 3.8e-32 | NA | linear | 1530 | 1370 |
| Faecalibacterium_prausnitzii | diagnosis | CD:dysbiosis_generaldysbiosis | diagnosisCD:dysbiosis_generaldysbiosis | -3.48 | 0.282 | 3.6e-35 | 3.52e-32 | 7.2e-35 | 3.8e-32 | NA | logistic | 1530 | 1370 |
| Clostridium_sp_AM49_4BH | diagnosis | CD | diagnosisCD | -0.68 | 0.344 | 0.108 | 0.197 | 2.31e-28 | 6.1e-26 | NA | linear | 1530 | 344 |
| Clostridium_sp_AM49_4BH | diagnosis | CD | diagnosisCD | -1.81 | 0.163 | 1.15e-28 | 4.51e-26 | 2.31e-28 | 6.1e-26 | NA | logistic | 1530 | 344 |
| Firmicutes_bacterium_AF16_15 | diagnosis | UC | diagnosisUC | -0.47 | 0.246 | 0.0924 | 0.174 | 1.4e-23 | 2.12e-21 | NA | linear | 1530 | 827 |
| Firmicutes_bacterium_AF16_15 | diagnosis | UC | diagnosisUC | -1.67 | 0.166 | 7.01e-24 | 1.71e-21 | 1.4e-23 | 2.12e-21 | NA | logistic | 1530 | 827 |
| Eubacterium_rectale | diagnosis | CD:dysbiosis_generaldysbiosis | diagnosisCD:dysbiosis_generaldysbiosis | -0.602 | 0.386 | 0.395 | 0.533 | 5.15e-23 | 6.79e-21 | NA | linear | 1530 | 1210 |
| Eubacterium_rectale | diagnosis | CD:dysbiosis_generaldysbiosis | diagnosisCD:dysbiosis_generaldysbiosis | -2.36 | 0.237 | 2.57e-23 | 5.59e-21 | 5.15e-23 | 6.79e-21 | NA | logistic | 1530 | 1210 |
| Dysosmobacter_welbionis | diagnosis | CD:dysbiosis_generaldysbiosis | diagnosisCD:dysbiosis_generaldysbiosis | 0.7 | 0.342 | 0.0316 | 0.075 | 7.7e-23 | 8.13e-21 | NA | linear | 1530 | 1190 |
| Dysosmobacter_welbionis | diagnosis | CD:dysbiosis_generaldysbiosis | diagnosisCD:dysbiosis_generaldysbiosis | -2.39 | 0.241 | 3.85e-23 | 6.84e-21 | 7.7e-23 | 8.13e-21 | NA | logistic | 1530 | 1190 |
| Blautia_faecis | diagnosis | CD:dysbiosis_generaldysbiosis | diagnosisCD:dysbiosis_generaldysbiosis | 1.03 | 0.474 | 0.0211 | 0.0532 | 5.45e-22 | 5.23e-20 | NA | linear | 1530 | 1120 |
| Blautia_faecis | diagnosis | CD:dysbiosis_generaldysbiosis | diagnosisCD:dysbiosis_generaldysbiosis | -2.42 | 0.249 | 2.72e-22 | 4.44e-20 | 5.45e-22 | 5.23e-20 | NA | logistic | 1530 | 1120 |
| Fusicatenibacter_saccharivorans | diagnosis | CD:dysbiosis_generaldysbiosis | diagnosisCD:dysbiosis_generaldysbiosis | 0.311 | 0.4 | 0.263 | 0.393 | 9.33e-22 | 8.21e-20 | NA | linear | 1530 | 1120 |
| Fusicatenibacter_saccharivorans | diagnosis | CD:dysbiosis_generaldysbiosis | diagnosisCD:dysbiosis_generaldysbiosis | -2.35 | 0.243 | 4.67e-22 | 7.01e-20 | 9.33e-22 | 8.21e-20 | NA | logistic | 1530 | 1120 |
| Lachnospira_sp_NSJ_43 | diagnosis | CD | diagnosisCD | -0.403 | 0.401 | 0.437 | 0.577 | 1.86e-20 | 1.51e-18 | NA | linear | 1530 | 296 |
| Lachnospira_sp_NSJ_43 | diagnosis | CD | diagnosisCD | -1.65 | 0.177 | 9.28e-21 | 1.21e-18 | 1.86e-20 | 1.51e-18 | NA | logistic | 1530 | 296 |
| Bacteroides_ovatus | diagnosis | CD:dysbiosis_generaldysbiosis | diagnosisCD:dysbiosis_generaldysbiosis | 0.278 | 0.429 | 0.316 | 0.454 | 2.25e-20 | 1.7e-18 | NA | linear | 1530 | 1180 |
| Bacteroides_ovatus | diagnosis | CD:dysbiosis_generaldysbiosis | diagnosisCD:dysbiosis_generaldysbiosis | -2.13 | 0.228 | 1.13e-20 | 1.37e-18 | 2.25e-20 | 1.7e-18 | NA | logistic | 1530 | 1180 |
| Clostridium_sp_AM49_4BH | diagnosis | UC | diagnosisUC | 0.136 | 0.357 | 0.877 | 0.931 | 5.16e-20 | 3.64e-18 | NA | linear | 1530 | 344 |
| Clostridium_sp_AM49_4BH | diagnosis | UC | diagnosisUC | -1.57 | 0.169 | 2.58e-20 | 2.97e-18 | 5.16e-20 | 3.64e-18 | NA | logistic | 1530 | 344 |
In the model above, we have converted dysbiosis_CD and dysbiosis_UC into dysbiosis in the variable dysbiosis_general and then specified an interaction between diagnosis and dysbiosis_general with diagnosis:dysbiosis_general. Since diagnosis has 3 levels itself (nonIBD, UC, CD), this will produce five terms (name column) for each feature:
diagnosisCD is the difference between non-dysbiotic CD and non-IBDdiagnosisUC is the difference between non-dysbiotic UC and non-IBDdiagnosisCD:dysbiosis_generaldysbiosis (the interaction of diagnosisCD and dysbiosis_generaldysbiosis) is the difference between non-dysbiotic CD and dysbiotic CDdiagnosisnonIBD:dysbiosis_generaldysbiosis (the interaction of diagnosisnonIBD and dysbiosis_generaldysbiosis) is the difference between dysbiotic and non-dysbiotic non-IBD. Note that these coefficients are all NA since no subjects labeled non-IBD were marked as being dysbiotic.diagnosisUC:dysbiosis_generaldysbiosis (the interaction of diagnosisUC and dysbiosis_generaldysbiosis) is the difference between non-dysbiotic UC and dysbiotic UCSince we have effectively fit the original model in a different way, this output table is equivalent to the original output table but with new names in the columns metadata, value, and name based on the interaction terms.
Another feature of MaAsLin 3 is the ability to test for level-versus-level differences in ordered predictors with contrast tests. Ordered predictors are categorical variables with a natural ordering such as cancer stage, consumption frequency of a dietary factor, or dosage group. Here, we assess how microbial abundances and prevalences are associated with eating red meat by including ordered(red_meat) in the formula. This will perform a contrast test for whether there is a difference between each pair of subsequent levels (e.g., “Yesterday, 3 or more times” versus “Yesterday, 1 to 2 times”) rather than whether there are differences between the levels and the baseline (e.g., “Yesterday, 3 or more times” versus “Not in the last 7 days”). The coefficient, standard error, and p-value all correspond to the difference between the level in value and the previous level. Ordered predictors should only be included as fixed effects (i.e., no ordered predictors as random effects etc.).
# Put the red meat consumption responses in order
metadata <- metadata %>%
mutate(red_meat = ifelse(
red_meat == 'No, I did not consume these products in the last 7 days',
'Not in the last 7 days',
red_meat) %>%
factor(levels = c('Not in the last 7 days',
'Within the past 4 to 7 days',
'Within the past 2 to 3 days',
'Yesterday, 1 to 2 times',
'Yesterday, 3 or more times'))
)
# Create the model with only non-IBD subjects
fit_out <- maaslin3(
input_data = taxa_table,
input_metadata = metadata[metadata$diagnosis == 'nonIBD',],
output = 'ordered_outputs',
formula = '~ ordered(red_meat) + antibiotics + age + reads',
plot_summary_plot = TRUE,
plot_associations = TRUE,
heatmap_vars = c('red_meat Within the past 4 to 7 days',
'red_meat Within the past 2 to 3 days',
'red_meat Yesterday, 1 to 2 times',
'red_meat Yesterday, 3 or more times'),
max_pngs = 30)If we look at the resulting heatmap, we can identify the Alistipes shahii prevalence association as potentially interesting since it increases in prevalence with more meat consumption in all but one level-versus-level comparison.
| feature | metadata | value | name | coef | stderr | pval_individual | qval_individual | pval_joint | qval_joint | error | model | N | N_not_zero |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Alistipes_shahii | red_meat | Within the past 4 to 7 days | red_meatWithin the past 4 to 7 days | 1.47 | 0.353 | 3.16e-05 | 0.00113 | 6.31e-05 | 0.00141 | NA | logistic | 405 | 266 |
| Alistipes_shahii | red_meat | Within the past 2 to 3 days | red_meatWithin the past 2 to 3 days | -0.67 | 0.316 | 0.0341 | 0.194 | 0.067 | 0.23 | NA | logistic | 405 | 266 |
| Alistipes_shahii | red_meat | Yesterday, 1 to 2 times | red_meatYesterday, 1 to 2 times | 1.07 | 0.291 | 0.000241 | 0.0064 | 0.000483 | 0.00788 | NA | logistic | 405 | 266 |
| Alistipes_shahii | red_meat | Yesterday, 3 or more times | red_meatYesterday, 3 or more times | 3.29 | 3.83 | 0.391 | 0.68 | 0.011 | 0.0688 | NA | logistic | 405 | 266 |
MaAsLin 3 can also test for group-wise differences in categorical predictors using an ANOVA or ANOVA-like procedure. Group-wise predictors are categorical variables with or without an ordering such as race, country, or consumption frequency of a dietary factor. When performing a group-wise difference test, we are not comparing any two particular levels; rather, we are investigating whether abundances and prevalences are the same for all levels. Therefore, no coefficient is returned, only a p-value. Here, we test whether there are differences in microbial abundances and prevalences across people with different levels of red meat consumption by including group(red_meat). Group-wise predictors should only be included as fixed effects (i.e., no group-wise predictors as random effects etc.).
fit_out <- maaslin3(
input_data = taxa_table,
input_metadata = metadata[metadata$diagnosis == 'nonIBD',],
output = 'group_outputs',
formula = '~ group(red_meat) + antibiotics + age + reads',
plot_summary_plot = TRUE,
plot_associations = TRUE,
heatmap_vars = c('red_meat Within the past 4 to 7 days',
'red_meat Within the past 2 to 3 days',
'red_meat Yesterday, 1 to 2 times',
'red_meat Yesterday, 3 or more times'),
max_pngs = 200)If we look at the same Alistipes shahii association from before, we see that no coefficient or standard error is returned for a group predictor, but the q-value is very low. This corroborates the observation earlier that there are differences in Alistipes shahii prevalence with red meat consumption.
| feature | metadata | value | name | coef | stderr | pval_individual | qval_individual | pval_joint | qval_joint | error | model | N | N_not_zero |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Alistipes_shahii | red_meat | NA | red_meat | NA | NA | 5.86e-09 | 5.88e-07 | 1.17e-08 | 7.29e-07 | NA | logistic | 405 | 266 |
We can also perform contrast testing in MaAsLin 3 to check whether a linear combination of the coefficients is significantly different from some constant on the right hand size (RHS). Specify a contrast matrix with column names matching the coefficient names and rows with the contrasts of interest. For each row \(C^T\), the hypothesis \(H_0:C^T\vec{\beta}=RHS\) will be tested. If no RHS is specified, a median test will be performed, and the median test RHS will be returned.
Below, we test whether the UC and CD diagnosis coefficients are the same and whether the UC and CD dysbiosis coefficients are the same. The contrast matrix can easily be extended to test any groups pairwise.
fit_out <- maaslin3(input_data = taxa_table,
input_metadata = metadata,
output = 'contrast_test_output',
formula = '~ diagnosis + dysbiosis_state +
antibiotics + age + reads',
plot_summary_plot = FALSE,
plot_associations = FALSE,
cores = 1)contrast_mat <- matrix(c(1, -1, 0, 0, 0, 0, 1, -1),
ncol = 4, nrow = 2, byrow = TRUE)
colnames(contrast_mat) <- c("diagnosisUC",
"diagnosisCD",
"dysbiosis_statedysbiosis_UC",
"dysbiosis_statedysbiosis_CD")
rownames(contrast_mat) <- c("diagnosis_test", "dysbiosis_test")
contrast_mat## diagnosisUC diagnosisCD dysbiosis_statedysbiosis_UC
## diagnosis_test 1 -1 0
## dysbiosis_test 0 0 1
## dysbiosis_statedysbiosis_CD
## diagnosis_test 0
## dysbiosis_test -1contrast_out <- maaslin_contrast_test(fits = fit_out$fit_data_abundance$fits,
contrast_mat = contrast_mat,
median_comparison = TRUE)
head(contrast_out, n = 20) %>%
knitr::kable() %>%
kableExtra::kable_styling("striped", position = 'center') %>%
kableExtra::scroll_box(width = "800px", height = "400px")| feature | test | coef | rhs | stderr | pval_individual | error | model |
|---|---|---|---|---|---|---|---|
| Phocaeicola_vulgatus | diagnosis_test | 0.1089748 | 0.1089748 | 0.2443056 | 1.0000000 | NA | abundance |
| Phocaeicola_vulgatus | dysbiosis_test | 0.3658063 | 0.3961497 | 0.9026559 | 0.9731889 | NA | abundance |
| Faecalibacterium_prausnitzii | diagnosis_test | 0.3506263 | 0.5303588 | 0.1259597 | 0.1538356 | NA | abundance |
| Faecalibacterium_prausnitzii | dysbiosis_test | 2.2499071 | 3.8075289 | 0.4748916 | 0.0010644 | NA | abundance |
| Bacteroides_uniformis | diagnosis_test | -0.1714861 | 0.0637664 | 0.1709981 | 0.1691449 | NA | abundance |
| Bacteroides_uniformis | dysbiosis_test | 2.4290291 | 4.2928010 | 0.6875146 | 0.0068034 | NA | abundance |
| Prevotella_copri_clade_A | diagnosis_test | 1.9979479 | 3.8654625 | 0.7520264 | 0.0136002 | NA | abundance |
| Prevotella_copri_clade_A | dysbiosis_test | 0.8652342 | 1.3337903 | 2.6151949 | 0.8579358 | NA | abundance |
| Bacteroides_stercoris | diagnosis_test | 0.1819607 | 0.2510259 | 0.2750066 | 0.8017688 | NA | abundance |
| Bacteroides_stercoris | dysbiosis_test | 2.9598746 | 5.4796559 | 1.4948726 | 0.0922512 | NA | abundance |
| Phocaeicola_dorei | diagnosis_test | 0.6128319 | 1.1089574 | 0.4890767 | 0.3107083 | NA | abundance |
| Phocaeicola_dorei | dysbiosis_test | 0.3971967 | 0.3971967 | 1.6972670 | 1.0000000 | NA | abundance |
| Bacteroides_ovatus | diagnosis_test | 0.6408062 | 1.0930804 | 0.1844777 | 0.0143660 | NA | abundance |
| Bacteroides_ovatus | dysbiosis_test | 1.3045581 | 2.1694292 | 0.8593975 | 0.3144465 | NA | abundance |
| Bacteroides_fragilis | diagnosis_test | -0.3317458 | 0.0755821 | 0.2672160 | 0.1278297 | NA | abundance |
| Bacteroides_fragilis | dysbiosis_test | -0.1072644 | 0.3718968 | 0.7859776 | 0.5422799 | NA | abundance |
| Eubacterium_rectale | diagnosis_test | 0.3292258 | 0.5147960 | 0.1685224 | 0.2710468 | NA | abundance |
| Eubacterium_rectale | dysbiosis_test | 1.8318224 | 3.1782115 | 0.8070358 | 0.0955136 | NA | abundance |
| Alistipes_putredinis | diagnosis_test | -0.8184856 | 0.0051087 | 0.2225484 | 0.0002286 | NA | abundance |
| Alistipes_putredinis | dysbiosis_test | 2.6075856 | 4.7650816 | 1.3397762 | 0.1076884 | NA | abundance |
MaAsLin 3 can also be run with a command line interface. For example, the first HMP2 analysis can be performed with the following command (the slashes may need to be removed):
./R/maaslin3.R \
inst/extdata/HMP2_taxonomy.tsv \
inst/extdata/HMP2_metadata.tsv \
command_line_output \
--formula='~ diagnosis + dysbiosis_state + antibiotics + age + reads' \
--reference='diagnosis,nonIBD;dysbiosis_state,none;antibiotics,No'./R/maaslin3.R).inst/extdata/HMP2_taxonomy.tsv is the path to your data (or features) fileinst/extdata/HMP2_metadata.tsv is the path to your metadata filecommand_line_output is the path to the folder to write the output| Tool | Models differential abundance | Models differential prevalence |
Models allowed | Accounts for compositionality |
Can use experimental absolute abundance information |
|---|---|---|---|---|---|
| MaAsLin 3 | Yes | Yes, for any model type | Any lme4 model (general mixed models), level-vs-level differences, group-wise differences, feature-specific covariates, contrast tests |
Yes | Yes |
| MaAsLin 2 | Yes | Only insofar as it affects abundance | Fixed and random effects (subset of general mixed models) |
No | No |
| ANCOM-BC2 | Yes | Only when completely absent in a group | Any lme4 model (general mixed models), level-vs-level differences, group-wise differences, contrast tests |
Yes | No |
| ALDEx2 | Yes | Only insofar as it affects abundance | Fixed effects |
Yes | No |
| LinDA | Yes | Only insofar as it affects abundance | Any lme4 model (general mixed models), group-wise differences, contrast tests |
Yes | No |
sessionInfo()## R Under development (unstable) (2025-01-20 r87609)
## Platform: x86_64-pc-linux-gnu
## Running under: Ubuntu 24.04.1 LTS
##
## Matrix products: default
## BLAS: /home/biocbuild/bbs-3.21-bioc/R/lib/libRblas.so
## LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.12.0 LAPACK version 3.12.0
##
## locale:
## [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
## [3] LC_TIME=en_GB LC_COLLATE=C
## [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
## [7] LC_PAPER=en_US.UTF-8 LC_NAME=C
## [9] LC_ADDRESS=C LC_TELEPHONE=C
## [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
##
## time zone: America/New_York
## tzcode source: system (glibc)
##
## attached base packages:
## [1] stats graphics grDevices utils datasets methods base
##
## other attached packages:
## [1] kableExtra_1.4.0 knitr_1.49 ggplot2_3.5.1 dplyr_1.1.4
## [5] maaslin3_0.99.4
##
## loaded via a namespace (and not attached):
## [1] Rdpack_2.6.2 pbapply_1.7-2
## [3] sandwich_3.1-1 rlang_1.1.5
## [5] magrittr_2.0.3 multcomp_1.4-28
## [7] matrixStats_1.5.0 compiler_4.5.0
## [9] mgcv_1.9-1 systemfonts_1.2.1
## [11] vctrs_0.6.5 stringr_1.5.1
## [13] pkgconfig_2.0.3 crayon_1.5.3
## [15] fastmap_1.2.0 XVector_0.47.2
## [17] labeling_0.4.3 rmarkdown_2.29
## [19] UCSC.utils_1.3.1 nloptr_2.1.1
## [21] ragg_1.3.3 purrr_1.0.2
## [23] xfun_0.50 cachem_1.1.0
## [25] GenomeInfoDb_1.43.4 jsonlite_1.8.9
## [27] DelayedArray_0.33.4 BiocParallel_1.41.0
## [29] parallel_4.5.0 R6_2.5.1
## [31] bslib_0.8.0 stringi_1.8.4
## [33] RColorBrewer_1.1-3 boot_1.3-31
## [35] numDeriv_2016.8-1.1 GenomicRanges_1.59.1
## [37] jquerylib_0.1.4 Rcpp_1.0.14
## [39] SummarizedExperiment_1.37.0 zoo_1.8-12
## [41] IRanges_2.41.2 Matrix_1.7-2
## [43] splines_4.5.0 tidyselect_1.2.1
## [45] rstudioapi_0.17.1 abind_1.4-8
## [47] yaml_2.3.10 TreeSummarizedExperiment_2.15.0
## [49] codetools_0.2-20 lmerTest_3.1-3
## [51] lattice_0.22-6 tibble_3.2.1
## [53] Biobase_2.67.0 treeio_1.31.0
## [55] withr_3.0.2 evaluate_1.0.3
## [57] survival_3.8-3 getopt_1.20.4
## [59] xml2_1.3.6 Biostrings_2.75.3
## [61] pillar_1.10.1 MatrixGenerics_1.19.1
## [63] stats4_4.5.0 reformulas_0.4.0
## [65] generics_0.1.3 S4Vectors_0.45.2
## [67] munsell_0.5.1 scales_1.3.0
## [69] tidytree_0.4.6 minqa_1.2.8
## [71] glue_1.8.0 lazyeval_0.2.2
## [73] tools_4.5.0 ggnewscale_0.5.0
## [75] lme4_1.1-36 fs_1.6.5
## [77] mvtnorm_1.3-3 grid_4.5.0
## [79] optparse_1.7.5 tidyr_1.3.1
## [81] ape_5.8-1 rbibutils_2.3
## [83] colorspace_2.1-1 SingleCellExperiment_1.29.1
## [85] nlme_3.1-167 GenomeInfoDbData_1.2.13
## [87] patchwork_1.3.0 cli_3.6.3
## [89] textshaping_1.0.0 S4Arrays_1.7.1
## [91] viridisLite_0.4.2 svglite_2.1.3
## [93] gtable_0.3.6 yulab.utils_0.2.0
## [95] logging_0.10-108 sass_0.4.9
## [97] digest_0.6.37 BiocGenerics_0.53.6
## [99] SparseArray_1.7.4 TH.data_1.1-3
## [101] farver_2.1.2 htmltools_0.5.8.1
## [103] lifecycle_1.0.4 httr_1.4.7
## [105] MASS_7.3-64# Clean-up
unlink('hmp2_output', recursive = TRUE)
unlink('spike_in_demo', recursive = TRUE)
unlink('total_scaling_demo', recursive = TRUE)
unlink('random_effects_output', recursive = TRUE)
unlink('interaction_output', recursive = TRUE)
unlink('ordered_outputs', recursive = TRUE)
unlink('group_outputs', recursive = TRUE)
unlink('contrast_test_output', recursive = TRUE)