---
title: "MaAsLin 3 User Manual"
author:
- name: William Nickols
  email: willnickols@g.harvard.edu
- name: Jacob Nearing
  email: nearing@broadinstitute.org
- name: Sagun Maharjan
  email: smaharjan@hsph.harvard.edu
output:
  html_document: default
  pdf_document: default
vignette: >
    %\VignetteIndexEntry{Manual}
    %\VignetteEngine{knitr::rmarkdown}
    %\VignetteEncoding{UTF-8}
---

## Introduction ##

[**MaAsLin 3**](http://huttenhower.sph.harvard.edu/MaAsLin3) is the next
generation of **M**a**A**s**L**in (**M**icrobiome **M**ultivariable
**A**ssociations with **L**inear 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

### Support ###
Check out the [MaAsLin 3
tutorial](https://github.com/biobakery/biobakery/wiki/MaAsLin3) for an
overview of analysis options and some example runs. 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 3
Forum](https://forum.biobakery.org/c/Downstream-analysis-and-statistics/maaslin)

--------------------------------------------

## Contents ##
- [Introduction](#introduction)
- [Support](#support)
- [Contents](#contents)
- [Requirements](#requirements)
- [Installation](#installation)
- [Running MaAsLin 3](#running-maaslin-3)
- [Input data](#input-data)
- [Output files](#output-files)
- [Run a demo](#run-a-demo)
    - [In R](#in-r)
    - [Plot revisions](#plot-revisions)
    - [Command line](#command-line)
    - [Contrast testing](#contrast-testing)
- [Options](#options)
    - [Required parameters](#required-parameters)
    - [Model formula](#model-formula)
    - [Analysis options](#analysis-options)
    - [Compositionality corrections](#compositionality-corrections)
    - [Plotting parameters](#plotting-parameters)
    - [Technical parameters](#technical-parameters)
- [Troubleshooting](#troubleshooting)

## Requirements ##

MaAsLin3 is an R package that can be run on the command line or as an R
function.

## Installation ##

#### Install using GitHub and devtools

The latest version of MaAsLin 3 can be installed from BiocManager.

```{r, eval=FALSE, cache = FALSE}
if (!require("BiocManager", quietly = TRUE))
    install.packages("BiocManager")

BiocManager::install("biobakery/maaslin3")
```

To compile vignettes yourself, specify `dependencies = TRUE`.

```{r, eval=TRUE, cache = FALSE, echo=FALSE}
for (lib in c('maaslin3', 'dplyr', 'ggplot2', 'knitr')) {
    suppressPackageStartupMessages(require(lib, character.only = TRUE))
}
```

## Running MaAsLin 3 ##

MaAsLin3 can be run from the command line or as an R function. Both
methods require the same arguments, have the same options, and use the
same default settings. 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.

### Input data ###

MaAsLin3 requires two input files.

1. Feature abundance data frame
    * Formatted with features as columns and samples as rows.
    * The transpose of this format is also okay.
    * Possible features include taxonomy or genes. These can be relative
    abundances or counts.
    * This can be a filepath to a tab-delimited file.
2. Metadata data frame
    * Formatted with variables as columns and samples as rows.
    * The transpose of this format is also okay.
    * Possible metadata include gender or age.
    * This can be a filepath to a tab-delimited file.

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.

To run MaAsLin 3, it is also necessary to specify 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*: The `formula` parameter should be set to any formula that
satisfies the `lme4` specifications: fixed effects, random effects,
interaction terms, 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, group
predictors, and strata variables can be included by including
`group(variable_name)`, `ordered(variable_name)`, and `strata(variable_name)`
respectively in the formula. Ordered and group
predictors should stand alone in the formula (i.e., no group predictors
in random effects). Only one strata variable can be included.
* *Vectors*: Alternatively, a vector of variable names can be supplied
to the parameters `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.**

### Output files ###

MaAsLin 3 generates two types of output files explained below: data and
visualizations. In addition, the object returned from `maaslin3`
contains all the data and results (see `?maaslin_fit`).

1. Data output files
    * ``all_results.tsv``
        * `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.
        * `null_hypothesis` is the value of the null hypothesis against which 
        the coefficient is tested (zero or the per-metadatum median).
        * `pval_individual` is the p-value of the individual association.
        * `qval_individual` is the 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.
        * `error` lists any errors from the model fitting.
        * `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.
    * ``significant_results.tsv``
        * This file is a subset of the results in the first file.
        * It only includes associations with joint or individual q-values less 
than or equal to the threshold.
    * ``features``
        * This folder includes the filtered, normalized, and transformed
        versions of the input feature table.
        * These steps are performed sequentially in the above order.
        * If an option is set such that a step does not change the data, the
        resulting table will still be output.
    * ``models_linear.rds`` and ``models_logistic.rds``
        * These files contain a list with every model fit object (`linear` for
        linear models, `logistic` for logistic models).
        * It will only be generated if `save_models` is set to TRUE.
    * ``residuals_linear.rds`` and ``residuals_logstic.rds``
        * These files contain a data frame with residuals for each feature.
    * ``fitted_linear.rds`` and ``fitted_logistic.rds``
        * These files contain a data frame with fitted values for each feature.
    * ``ranef_linear.rds`` and ``ranef_logistic.rds``
        * These files contain a data frame with extracted random effects for
        each feature (when random effects are specified).
    * ``maaslin3.log``
        * This file contains all log information for the run.
        * It includes all settings, warnings, errors, and steps run.
2. Visualization output files
    * ``summary_plot.pdf``
        * This file contain a combined coefficient plot and heatmap of the most
        significant associations. In the heatmap, one star indicates the
        individual q-value is below the parameter `max_significance`, and two
        stars indicate the individual q-value is below `max_significance`
        divided by 10.
    * ``association_plots/[metadatum]/[association]/
[metadatum]_[feature]_[association].png``
        * A plot is generated for each significant association up to `max_pngs`.
        * Scatter plots are used for continuous metadata abundance associations.
        * Box plots are used for categorical data abundance associations.
        * Box plots are used for continuous data prevalence associations.
        * Grids are used for categorical data prevalence associations.
        * Data points plotted are after filtering, normalization, and
        transformation so that the scale in the plot is the scale that was used
        in fitting.

At 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.

#### Diagnostics

1. When warnings or errors are thrown during the fitting process, they are 
recorded in the `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.
2. Despite the error checking, significant results could still result from poor 
model fits. These can usually be diagnosed with the visuals in the 
`association_plots` directory. 
    * Any significant abundance associations with a categorical variable should 
    usually have **at least 10 observations in each category**.
    * Significant prevalence associations with categorical variables should 
    also have **at least 10 samples in which the feature was present and at 
    least 10 samples in which it was absent for each significant category**. 
    * Significant abundance associations with continuous metadata should be 
    checked visually for influential outliers.
3. The q-values are FDR corrected over all abundance or prevalence 
relationships (separately), so it may be preferable to FDR correct just the 
p-values from the variables of interest. This can reduce false positives when 
there are many significant but uninteresting associations (e.g., many read 
depth associations).
4. There are also a few rules of thumb to keep in mind.
    * Models should ideally have about 10 times as many samples (all samples 
    for logistic fits, non-zero samples for linear fits) as covariate terms 
    (all continuous variables plus all categorical variable levels).
    * Coefficients (effect sizes) larger than about 15 in absolute value are 
    usually suspect unless very small unstandardized predictors are being 
    included. (A coefficient of 15 corresponds to a fold change >30000!). If 
    you encounter such coefficients, check that (1) no error was thrown, (2) 
    the plots look reasonable, (3) a sufficient number of samples were 
    used in fitting, (4) the q-value is significant, (5) the metadata are not 
    highly collinear, and (6) the random effects are plausible.

### Run a demo ###

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.

#### In R ####

The following code identifies associations between patient metadata and
microbial species in the HMP2 cohort.

```{R, echo = TRUE, results = 'hide', warning = FALSE, cache = FALSE}
# Read abundance table
taxa_table_name <- system.file("extdata", "HMP2_taxonomy.tsv",
                                package = "maaslin3")
taxa_table <- read.csv(taxa_table_name, sep = '\t', row.names = 1)

# Read metadata table
metadata_name <- system.file("extdata", "HMP2_metadata.tsv",
                                package = "maaslin3")
metadata <- read.csv(metadata_name, sep = '\t', row.names = 1)

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'))

# Fit models
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 = 20)
```

The `maaslin3` wrapper function runs all steps at once, but equivalent
results would be produced from running one step at a time:

```{r, eval=FALSE}
maaslin_log_arguments(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 = 20)

read_data_list <- maaslin_read_data(taxa_table,
                                    metadata)
read_data_list <- maaslin_reorder_data(read_data_list$data,
                                    read_data_list$metadata)

data <- read_data_list$data
metadata <- read_data_list$metadata

formulas <- maaslin_check_formula(data,
                                metadata,
                                '~ diagnosis + dysbiosis_state +
                                    antibiotics + age + reads')

formula <- formulas$formula
random_effects_formula <- formulas$random_effects_formula

normalized_data = maaslin_normalize(data,
                                    output = 'hmp2_output',
                                    normalization = 'TSS')

filtered_data = maaslin_filter(normalized_data,
                            output = 'hmp2_output')

transformed_data = maaslin_transform(filtered_data,
                                    output = 'hmp2_output',
                                    transform = 'LOG')

standardized_metadata = maaslin_process_metadata(metadata,
                                                formula,
                                                standardize = TRUE)

maaslin_results = maaslin_fit(filtered_data,
                            transformed_data,
                            standardized_metadata,
                            formula,
                            random_effects_formula,
                            data = data,
                            median_comparison_abundance = TRUE,
                            median_comparison_prevalence = FALSE)

maaslin_write_results(output = 'hmp2_output',
                    maaslin_results$fit_data_abundance,
                    maaslin_results$fit_data_prevalence,
                    random_effects_formula,
                    max_significance = 0.1)

# Invisible to avoid dumping plots
invisible(maaslin_plot_results(output = 'hmp2_output',
                            transformed_data,
                            metadata,
                            maaslin_results$fit_data_abundance,
                            maaslin_results$fit_data_prevalence,
                            normalization = "TSS",
                            transform = "LOG",
                            median_comparison_abundance = TRUE,
                            median_comparison_prevalence = FALSE,
                            max_significance = 0.1,
                            max_pngs = 20))
```

#### Replotting

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`.

```{R, echo = TRUE, results = 'hide', warning = FALSE, cache = FALSE}
# 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
colnames(taxa_table) <- gsub('_', ' ', colnames(taxa_table)) %>%
    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
colnames(metadata) <-
    case_when(colnames(metadata) == 'age' ~ 'Age',
            colnames(metadata) == 'antibiotics' ~ 'Abx',
            colnames(metadata) == 'diagnosis' ~ 'Diagnosis',
            colnames(metadata) == 'dysbiosis_state' ~ 'Dysbiosis',
            colnames(metadata) == 'reads' ~ 'Read depth',
            TRUE ~ colnames(metadata))
metadata <- metadata %>%
    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,
    normalization = "TSS",
    transform = "LOG",
    median_comparison_abundance = TRUE,
    median_comparison_prevalence = FALSE,
    max_significance = 0.1,
    coef_plot_vars=coef_plot_vars,
    heatmap_vars=heatmap_vars,
    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. (Make sure to set `include=T` to view
the plots when knitting the Rmd.)
```{r, out.width='100%', echo=FALSE, cache = FALSE, include=FALSE}
knitr::include_graphics("hmp2_output/figures/summary_plot.png")
```

In the association plots below, the taxa and metadata have been renamed to be
consistent with the results file from earlier. **To see the plots, set 
`max_pngs = 250` above and set `eval=TRUE` in this chunk.**
```{r, echo=FALSE, fig.show='hold', cache = FALSE, include=FALSE, eval=FALSE}
prefix <- "hmp2_output/figures/association_plots"
plot_vec <- c("/Age/linear/Age_Enterocloster clostridioformis_linear.png",
            "/Dysbiosis/linear/Dysbiosis_Escherichia coli_linear.png",
            "/Age/logistic/Age_Bifidobacterium longum_logistic.png",
            paste0("/Dysbiosis/logistic/",
                "Dysbiosis_Faecalibacterium prausnitzii_logistic.png"
                ))
knitr::include_graphics(paste0(prefix, plot_vec))
```

#### Contrast testing

We can also perform contrast testing in MaAsLin 3 to check whether a linear
combination of the coefficients is significantly different from some constant. 
Specify a contrast matrix
with column names matching the coefficient names and rows with the contrasts
of interest. A separate contrast test will be performed for each row.

```{R, echo = TRUE, results = 'hide', warning = FALSE, cache = FALSE}
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
                            
maaslin_contrast_test(maaslin3_fit = fit_out,
                        contrast_mat = contrast_mat)
```


#### Command line ####

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, engine = 'bash', eval = FALSE, cache = FALSE}
./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'
```

* Make sure to provide the full path to the MaAsLin3 executable (i.e.
`./R/maaslin3.R`).
* In the demo command:
    * ``inst/extdata/HMP2_taxonomy.tsv`` is the path to your data (or
    features) file
    * ``inst/extdata/HMP2_metadata.tsv`` is the path to your metadata file
    * ``command_line_output`` is the path to the folder to write the output

### Options ###

From the command line, the following command will print the list of
MaAsLin 3 options and default settings:

```$ ./R/maaslin3.R --help```

When running MaAsLin 3 in R, the manual page for each function (e.g.,
`?maaslin3`) will show the available options and default settings. For
both, the options and settings are as follows:

#### Required parameters ####

* `input_data`: A data frame of feature abundances or read counts or a
filepath to a tab-delimited file with abundances. It should be formatted
with features as columns and samples as rows (or the transpose). The
column and row names should be the feature names and sample names
respectively.
* `input_metadata`: A data frame of per-sample metadata or a filepath to
a tab-delimited file with metadata. It should be formatted with
variables as columns and samples as rows (or the transpose). The column
and row names should be the variable names and sample names
respectively.
* `output`: The output folder to write results.

#### Model formula ####

* `formula`: A formula in `lme4` format. Random effects, interactions,
and functions of the metadata can be included (note that these functions
will be applied after standardization if `standardize = TRUE`). Group,
ordered, and strata variables can be specified as:
`group(grouping_variable)`, `ordered(ordered_variable)`, and
`strata(strata_variable)`. The other variable options below will not be
considered if a formula is set.
* `fixed_effects`: A vector of variable names to be included as fixed
effects.
    * Fixed effects models are fit with `lm` (linear) or `glm` (logistic).
* `reference`: For a variable with more than two levels supplied with
`fixed_effects`, the factor to use as a reference provided as a string
of 'variable,reference' semi-colon delimited for multiple variables.
* `random_effects`: A vector of variable names to be included as random
intercepts. **Random intercept models may produce poor model fits when
there are fewer than 5 observations per group.** In these scenarios,
per-group fixed effects should be used and subsequently filtered out.
(See `strata_effects` as well.)
    * Random effects models are fit with `lmer` (linear) and `glmer`
(logistic), and the significance tests come from `lmerTest` and `glmer`
respectively.
* `group_effects`: A factored categorical variable to be included for
group testing. An ANOVA-style test will be performed to assess whether
any of the variable's levels are significant, and no coefficients or
individual p-values will be returned.
    * Tests are performed with the `anova` function's `LRT` option (logistic
fixed and mixed effects), the `anova` function's F test (linear fixed
effects), or `lmerTest::contest` (linear mixed effects).
* `ordered_effects`: A factored categorical variable to be included.
Consecutive levels will be tested for significance against each other
with contrast tests, and the resulting associations will correspond to
effect sizes, standard errors, and significances of each level versus
the previous.
    * Contrast tests are performed with `multcomp::glht` (fixed effects and
logistic mixed effects) and `lmerTest::contest` (linear mixed effects).
* `strata_effects`: A single grouping variable to be included in matched
case-control studies. If a strata variable is included, no random
effects can be included. When a strata variable is included, a
conditional logistic regression will be run to account for the strata.
The abundance model will be run with a random intercept in place of the
strata. Strata can include more than two observations per group. Only
variables that differ within the groups can be tested. In general, strata
effects are not recommended except for advanced users. Fixed or random
intercepts are recommended instead.

#### Feature specific covariates ####
Particularly for use in metatranscriptomics workflows, a table of
feature-specific covariates can be included. A feature's covariates will
be included like a fixed effect metadatum when fitting the model for
that feature. The covariate's name does not need to be included in the
formula.

* `feature_specific_covariate`: A table of feature-specific covariates
or a filepath to a tab-delimited file with feature-specific covariates.
It should be formatted with features as columns and samples as rows (or
the transpose). The row names and column names should be the same as
those of the `input_data`: the column and row names should be the
feature names and sample names respectively. Typically, this table should be
generated by `preprocess_dna_mtx` or `preprocess_taxa_mtx` first.
* `feature_specific_covariate_name`: The name for the feature-specific
covariates when fitting the models.
* `feature_specific_covariate_record`: Whether to keep the
feature-specific covariates in the outputs when calculating p-values,
writing results, and displaying plots.

#### Analysis options ####

* `min_abundance` (default `0`): Features with abundances of more than
`min_abundance` in more than `min_prevalence` of the samples will be 
included for analysis. The threshold is applied after normalization and before
transformation.
* `min_prevalence` (default `0`): See above.
* `max_prevalence` (default `1.01`): Features with abundances more than
`min_abundance` in fewer than `max_prevalence` of the samples will be 
included for analysis. The threshold is applied after normalization and
before transformation.
* `zero_threshold` (default `0`): Abundances less than or equal to
`zero_threshold` will be treated as zeros. This is primarily to be used
when the abundance table has likely low-abundance false positives.
* `min_variance` (default `0`): Features with abundance variances less
than or equal to `min_variance` will be dropped. This is primarily used
for dropping features that are entirely zero.
* `max_significance` (default `0.1`): The FDR corrected q-value
threshold for significance used in selecting which associations to write
as significant and to plot.
* `normalization` (default `TSS`): The normalization to apply to the
features before transformation and analysis. The option `TSS` (total-sum
scaling) is recommended, but `CLR` (centered log ratio) and `NONE` can
also be used.
* `transform` (default `LOG`, base 2): The transformation to apply to
the features after normalization and before analysis. The option `LOG`
is recommended, but `PLOG` (pseudo-log with a pseudo-count of half the 
dataset minimum non-zero abundance replacing zeros, particularly for
metabolomics data) and `NONE` can also be used.
* `correction` (default `BH`): The correction to obtain FDR-corrected
q-values from raw p-values. Any valid options for `p.adjust` can be
used.
* `standardize` (default `TRUE`): Whether to apply z-scores to
continuous metadata variables so they are on the same scale. This is
recommended in order to compare coefficients across metadata variables,
but note that functions of the metadata specified in the `formula` will
apply after standardization.
* `warn_prevalence` (default `TRUE`): Warn when prevalence associations 
are likely induced by abundance associations. This requires re-fitting the 
linear models on the TSS log-transformed data. A prevalence coefficient is
flagged if its corresponding abundance coefficient is significantly different
from 0, of the same sign, and larger in absolute value.
* `small_random_effects` (default `FALSE`): Automatically replace random 
effects with fixed effects in the logistic prevalence model to handle low 
numbers of observations per group.
* `bypass_small_group_warning` (default `FALSE`): When `TRUE`, bypass the 
warning that is normally added to results when there are fewer than 4 average 
observations per random effect group.
* `augment` (default `TRUE`): To avoid linear separability in the
logistic regression, at each input data point, add an extra 0 and an
extra 1 observation weighted as the number of predictors divided by two
times the number of data points. This is almost always recommended to
avoid linear separability while having a minor effect on fit
coefficients otherwise.
* `evaluate_only` (default `NULL`): To fit only the abundance or only the
prevalence models, `evaluate_only` can be set to `abundance` or `prevalence`.

#### Compositionality corrections ####

##### Absolute abundance

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:
$$\textrm{Absolute abundance microbe}=(\textrm{Total absolute
abundance})\cdot(\textrm{Relative abundance microbe})$$

##### Compositionality corrections continued

* `unscaled_abundance`: A data frame with a single column of absolute
abundances or a filepath to such a tab-delimited file. The row names
should match the names of the samples in `input_data` and
`input_metadata`. When using spike-ins, the single column should have
the same name as one of the features in `input_data`, and the values
should correspond to the absolute quantity of the spike-in. For example,
if the same spike-in quantity is used in each sample, the entire column
can be set to 1. When using total abundance scaling, the single column
should have the name 'total', and the values should correspond to the
total abundance of each sample. In both cases,
`median_comparison_abundance` should be set to `FALSE` since the
spike-in or total abundance normalization accounts for compositionality.

Alternatively, if the `input_data` abundances have already been scaled
to be absolute abundances, the user should set `normalization = NONE`
and `median_comparison_abundance = FALSE` and not include anything for
`unscaled_abundance`. Then, the absolute abundances will be log
transformed, and models will be fit on those values directly.

##### Median comparisons

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 if only
one feature has a positive association with a metadatum on the absolute
scale (cell count), the other features will have apparent negative
associations with that metadatum on the relative scale (proportion of
the community) because relative abundances must sum to 1. More
generally, associations on the relative scale are not necessarily the
same as the associations on the absolute scale in magnitude or sign, so
**testing against zero on the relative scale is not equivalent to
testing against zero on the absolute scale**. When testing associations
on the relative scale, the coefficients should be tested against 0
(median comparison off). However, since these tests do not correspond to
tests for associations on the absolute scale, we instead use a test
against the median, which can enable some inference on the absolute
scale. There are two interpretations of this test for absolute abundance
associations:

1. In linear models without sparsity (or with sparsity under some assumptions), 
if two features' associations with a particular
metadatum on the log *absolute* scale differ by some value $d$, the
features' associations with that metadatum on the log *relative* scale
(total-sum scaling) will also differ by $d$. That is, the absolute and
relative coefficients for a particular feature-metadatum association are
different, but **the ordering of the relative coefficients is the same
as the ordering of the absolute coefficients for a metadatum, and the
difference between two coefficients on the relative scale is the same as
the difference between the corresponding coefficients on the absolute
scale**. Therefore, the test against the relative coefficient median can
always be interpreted as "a test of whether a particular association is
significantly different from the typical (median) association on the
absolute scale."
2. Under the assumption that at least half the features are not changing
on the absolute scale, the median true absolute coefficient is 0, so
this can be interpreted as a test of whether the feature has a non-zero
association on the absolute scale.

By contrast, sparsity should be less affected by compositionality since
a feature should still be present even if another increases or decreases
in abundance. (Note that, because the read depth is finite, this might
not always be true in practice.) Therefore,
`median_comparison_prevalence` is off by default, but it can be turned
on if the user is interested in testing whether a particular prevalence
association is significantly different from the typical prevalence
association.

In both cases, if the tested coefficient is within
`median_comparison_[abundance/prevalence]_threshold` of the median, it
will automatically receive a p-value of 1. This is based on the idea
that the association might be statistically significantly different but
not substantially different from the median and therefore is likely
still a result of compositionality.

To conclude:

* `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 set to `TRUE` in which
case the median will be subtracted.
* `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.

##### Compositionality corrections continued

* `median_comparison_abundance` (default `TRUE`): Test abundance
coefficients against a null value corresponding to the median
coefficient for a metadata variable across the features. Otherwise, test
against 0. This is recommended for relative abundance data but should
not be used for absolute abundance data.
* `median_comparison_prevalence` (default `FALSE`): Test prevalence
coefficients against a null value corresponding to the median
coefficient for a metadata variable across the features. Otherwise, test
against 0. This is only recommended if the analyst is interested in how
feature prevalence associations compare to each other or if there is
likely strong compositionality-induced sparsity.
* `median_comparison_abundance_threshold` (default `0`): Coefficients
within `median_comparison_abundance_threshold` of the median association
will automatically be counted as insignificant (p-value set to 1) since
they likely represent compositionality-induced associations. This
threshold will be divided by the metadata variable's standard deviation
if the metadatum is continuous to ensure the threshold applies to the
right scale.
* `median_comparison_prevalence_threshold` (default `0`): Same as
`median_comparison_abundance_threshold` but applied to the prevalence
associations.
* `subtract_median` (default `FALSE`): Subtract the median from the 
coefficients.

#### Plotting parameters ####

* `plot_summary_plot` (default `TRUE`): Generate a summary plot of
significant associations.
* `summary_plot_first_n` (default `25`): Include the top
`summary_plot_first_n` features with significant associations.
* `coef_plot_vars`: Vector of variable names to be used in the
coefficient plot section of the summary plot. Continuous variables
should match the metadata column name, and categorical variables should
be of the form `"[variable] [level]"`.

By default, the (up to) two metadata variables with the most significant
associations will be plotted in the coefficient plot, and the rest will
be plotted in the heatmap. Because predicting the output variable names
can be tricky, it is recommended to first run `maaslin3` without setting
`coef_plot_vars` or `heatmap_vars`, look at the names of the variables
in the summary plot, and then rerun with
`maaslin_plot_results_from_output` after updating `coef_plot_vars` and
`heatmap_vars` with the desired variables.
* `heatmap_vars`: Vector of variable names to be used in the heatmap
section of the summary plot. Continuous variables should match the
metadata column name, and categorical variables should be of the form
`"[variable] [level]"`.
* `plot_associations` (default `TRUE`): Whether to generate plots for
significant associations.
* `max_pngs` (default `30`): The top `max_pngs` significant associations
will be plotted.

#### Technical/miscellaneous parameters ####

* `save_models` (default `FALSE`): Whether to return the fit models and
save them to an RData file.
* `verbosity` (default `'FINEST'`): The level of verbosity for MaAsLin 3's
internal logger.
* `save_plots_rds` (default `FALSE`): Whether to save the plots as RDS files.
* `assay.type` (default `1`): A string or index to select the assay when using
a `SummarizedExperiment` object.
* Enable parallelization by creating background 
[daemons](https://mirai.r-lib.org/reference/daemons.html) using 
[mirai](https://mirai.r-lib.org/articles/mirai.html) e.g. call 
`mirai::daemons(2)` before running `maaslin3()` to use two cores.

## Tool comparison ##

| 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 |

## Troubleshooting ##

1. Question: When I run from the command line I see the error
``maaslin3.R: command not found``. How do I fix this?
    * Answer: Provide the full path to the executable when running
    maaslin3.R
2. Question: When I run as a function I see the error ``Error in
library(maaslin3): there is no package called 'maaslin3'``. How do I fix
this?
    * Answer: Install the R package and then try loading the library again.

```{R}
sessionInfo()
```

```{R}
# Clean-up generated outputs
unlink('hmp2_output', recursive = TRUE)
```
