MaAsLin2 User Manual

MaAsLin2 is the next generation of MaAsLin (Microbiome Multivariable Association with Linear Models).

MaAsLin2 is comprehensive R package for efficiently determining multivariable association between clinical metadata and microbial meta-omics features. MaAsLin2 relies on general linear models to accommodate most modern epidemiological study designs, including cross-sectional and longitudinal, along with a variety of filtering, normalization, and transform methods.

If you use the MaAsLin2 software, please cite our manuscript:

Mallick H, Rahnavard A, McIver LJ, Ma S, Zhang Y, Nguyen LH, Tickle TL, Weingart G, Ren B, Schwager EH, Chatterjee S, Thompson KN, Wilkinson JE, Subramanian A, Lu Y, Waldron L, Paulson JN, Franzosa EA, Bravo HC, Huttenhower C (2021). Multivariable Association Discovery in Population-scale Meta-omics Studies. PLoS Computational Biology, 17(11):e1009442.

Check out the MaAsLin 2 tutorial for an overview of analysis options.

If you have questions, please direct it to :
MaAsLin2 Forum
Google Groups (Read only)


Description

MaAsLin2 finds associations between microbiome multi-omics features and complex metadata in population-scale epidemiological studies. The software includes multiple analysis methods (with support for multiple covariates and repeated measures), filtering, normalization, and transform options to customize analysis for your specific study.

Requirements

MaAsLin2 is an R package that can be run on the command line or as an R function.

Installation

MaAsLin2 can be run from the command line or as an R function. If only running from the command line, you do not need to install the MaAsLin2 package but you will need to install the MaAsLin2 dependencies.

From command line

  1. Download the source: MaAsLin2.tar.gz
  2. Decompress the download:
    • $ tar xzvf maaslin2.tar.gz
  3. Install the Bioconductor dependencies edgeR and metagenomeSeq.
  4. Install the CRAN dependencies:
    • $ R -q -e "install.packages(c('lmerTest','pbapply','car','dplyr','vegan','chemometrics','ggplot2','pheatmap','hash','logging','data.table','glmmTMB','MASS','cplm','pscl'), repos='http://cran.r-project.org')"
  5. Install the MaAsLin2 package (only r,equired if running as an R function):
    • $ R CMD INSTALL maaslin2

From R

Install Bioconductor and then install Maaslin2

if(!requireNamespace("BiocManager", quietly = TRUE))
    install.packages("BiocManager")
BiocManager::install("Maaslin2")

How to Run

MaAsLin2 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.

Input Files

MaAsLin2 requires two input files.

  1. Data (or features) file
    • This file is tab-delimited.
    • Formatted with features as columns and samples as rows.
    • The transpose of this format is also okay.
    • Possible features in this file include taxonomy or genes.
  2. Metadata file
    • This file is tab-delimited.
    • Formatted with features as columns and samples as rows.
    • The transpose of this format is also okay.
    • Possible metadata in this file include gender or age.

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.

NOTE: If running MaAsLin2 as a function, the data and metadata inputs can be of type data.frame instead of a path to a file.

Output Files

MaAsLin2 generates two types of output files: data and visualization.

  1. Data output files
    • all_results.tsv
      • This includes the same data as the data.frame returned.
      • This file contains all results ordered by increasing q-value.
      • The first columns are the metadata and feature names.
      • The next two columns are the value and coefficient from the model.
      • The next column is the standard deviation from the model.
      • The N column is the total number of data points.
      • The N.not.zero column is the total of non-zero data points.
      • The pvalue from the calculation is the second to last column.
      • The qvalue is computed with p.adjust with the correction method.
    • significant_results.tsv
      • This file is a subset of the results in the first file.
      • It only includes associations with q-values <= 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.rds
      • This file contains a list with every model fit object.
      • It will only be generated if save_models is set to TRUE.
    • residuals.rds
      • This file contains a data frame with residuals for each feature.
    • fitted.rds
      • This file contains a data frame with fitted values for each feature.
    • ranef.rds
      • This file contains a data frame with extracted random effects for each feature (when random effects are specified).
    • maaslin2.log
      • This file contains all log information for the run.
      • It includes all settings, warnings, errors, and steps run.
  2. Visualization output files
    • heatmap.pdf
      • This file contains a heatmap of the significant associations.
    • [a-z/0-9]+.pdf
      • A plot is generated for each significant association.
      • Scatter plots are used for continuous metadata.
      • Box plots are for categorical data.
      • Data points plotted are after filtering but prior to normalization and transform.

Run a Demo

Example input files can be found in the inst/extdata folder of the MaAsLin2 source. The files provided were generated from the HMP2 data which can be downloaded from https://ibdmdb.org/ .

HMP2_taxonomy.tsv: is a tab-demilited file with species as columns and samples as rows. It is a subset of the taxonomy file so it just includes the species abundances for all samples.

HMP2_metadata.tsv: is a tab-delimited file with samples as rows and metadata as columns. It is a subset of the metadata file so that it just includes some of the fields.

Command line

$ Maaslin2.R --fixed_effects="diagnosis,dysbiosisnonIBD,dysbiosisUC,dysbiosisCD,antibiotics,age" --random_effects="site,subject" --standardize=FALSE inst/extdata/HMP2_taxonomy.tsv inst/extdata/HMP2_metadata.tsv demo_output

  • Make sure to provide the full path to the MaAsLin2 executable (ie ./R/Maaslin2.R).
  • In the demo command:
    • HMP2_taxonomy.tsv is the path to your data (or features) file
    • HMP2_metadata.tsv is the path to your metadata file
    • demo_output is the path to the folder to write the output

In R

library(Maaslin2)
input_data <- system.file(
    'extdata','HMP2_taxonomy.tsv', package="Maaslin2")
input_metadata <-system.file(
    'extdata','HMP2_metadata.tsv', package="Maaslin2")
fit_data <- Maaslin2(
    input_data, input_metadata, 'demo_output',
    fixed_effects = c('diagnosis', 'dysbiosisnonIBD','dysbiosisUC','dysbiosisCD', 'antibiotics', 'age'),
    random_effects = c('site', 'subject'),
    reference = "diagnosis,nonIBD",
    standardize = FALSE)
## [1] "Creating output folder"
## [1] "Creating output feature tables folder"
## [1] "Creating output fits folder"
## [1] "Creating output figures folder"
## 2024-12-18 19:17:17.308654 INFO::Writing function arguments to log file
## 2024-12-18 19:17:17.350277 INFO::Verifying options selected are valid
## 2024-12-18 19:17:17.381246 INFO::Determining format of input files
## 2024-12-18 19:17:17.383281 INFO::Input format is data samples as rows and metadata samples as rows
## 2024-12-18 19:17:17.390744 INFO::Formula for random effects: expr ~ (1 | site) + (1 | subject)
## 2024-12-18 19:17:17.392658 INFO::Formula for fixed effects: expr ~  diagnosis + dysbiosisnonIBD + dysbiosisUC + dysbiosisCD + antibiotics + age
## 2024-12-18 19:17:17.396841 INFO::Filter data based on min abundance and min prevalence
## 2024-12-18 19:17:17.400923 INFO::Total samples in data: 1595
## 2024-12-18 19:17:17.402336 INFO::Min samples required with min abundance for a feature not to be filtered: 159.500000
## 2024-12-18 19:17:17.407266 INFO::Total filtered features: 0
## 2024-12-18 19:17:17.408848 INFO::Filtered feature names from abundance and prevalence filtering:
## 2024-12-18 19:17:17.417114 INFO::Total filtered features with variance filtering: 0
## 2024-12-18 19:17:17.418783 INFO::Filtered feature names from variance filtering:
## 2024-12-18 19:17:17.420171 INFO::Running selected normalization method: TSS
## 2024-12-18 19:17:18.723301 INFO::Bypass z-score application to metadata
## 2024-12-18 19:17:18.725045 INFO::Running selected transform method: LOG
## 2024-12-18 19:17:18.745905 INFO::Running selected analysis method: LM
## 2024-12-18 19:17:19.125474 INFO::Fitting model to feature number 1, Bifidobacterium.adolescentis
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:19.568275 INFO::Fitting model to feature number 2, Bifidobacterium.bifidum
## 2024-12-18 19:17:19.971238 INFO::Fitting model to feature number 3, Bifidobacterium.longum
## 2024-12-18 19:17:20.131384 INFO::Fitting model to feature number 4, Bifidobacterium.pseudocatenulatum
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:20.278849 INFO::Fitting model to feature number 5, Collinsella.aerofaciens
## 2024-12-18 19:17:20.443527 INFO::Fitting model to feature number 6, Bacteroides.caccae
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:20.640889 INFO::Fitting model to feature number 7, Bacteroides.cellulosilyticus
## 2024-12-18 19:17:20.826428 INFO::Fitting model to feature number 8, Bacteroides.dorei
## 2024-12-18 19:17:21.044966 INFO::Fitting model to feature number 9, Bacteroides.eggerthii
## 2024-12-18 19:17:21.198721 INFO::Fitting model to feature number 10, Bacteroides.faecis
## 2024-12-18 19:17:21.363586 INFO::Fitting model to feature number 11, Bacteroides.finegoldii
## boundary (singular) fit: see help('isSingular')
## Feature Bacteroides.finegoldii : simpleWarning: Model failed to converge with 1 negative eigenvalue: -7.9e+01
## 2024-12-18 19:17:21.475555 WARNING::Fitting problem for feature 11 a warning was issued
## boundary (singular) fit: see help('isSingular')
## Warning: Model failed to converge with 1 negative eigenvalue: -7.9e+01
## 2024-12-18 19:17:21.622795 INFO::Fitting model to feature number 12, Bacteroides.fragilis
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:21.827268 INFO::Fitting model to feature number 13, Bacteroides.intestinalis
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:21.959372 INFO::Fitting model to feature number 14, Bacteroides.massiliensis
## 2024-12-18 19:17:22.08642 INFO::Fitting model to feature number 15, Bacteroides.ovatus
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:22.246232 INFO::Fitting model to feature number 16, Bacteroides.salyersiae
## 2024-12-18 19:17:22.391573 INFO::Fitting model to feature number 17, Bacteroides.stercoris
## 2024-12-18 19:17:22.51969 INFO::Fitting model to feature number 18, Bacteroides.thetaiotaomicron
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:22.641719 INFO::Fitting model to feature number 19, Bacteroides.uniformis
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:22.778062 INFO::Fitting model to feature number 20, Bacteroides.vulgatus
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:22.958628 INFO::Fitting model to feature number 21, Bacteroides.xylanisolvens
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:23.168843 INFO::Fitting model to feature number 22, Bacteroidales.bacterium.ph8
## 2024-12-18 19:17:23.386401 INFO::Fitting model to feature number 23, Barnesiella.intestinihominis
## 2024-12-18 19:17:23.545629 INFO::Fitting model to feature number 24, Coprobacter.fastidiosus
## 2024-12-18 19:17:23.699649 INFO::Fitting model to feature number 25, Odoribacter.splanchnicus
## 2024-12-18 19:17:23.921287 INFO::Fitting model to feature number 26, Parabacteroides.distasonis
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:24.123307 INFO::Fitting model to feature number 27, Parabacteroides.goldsteinii
## 2024-12-18 19:17:24.326486 INFO::Fitting model to feature number 28, Parabacteroides.merdae
## 2024-12-18 19:17:24.514063 INFO::Fitting model to feature number 29, Parabacteroides.unclassified
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:24.651245 INFO::Fitting model to feature number 30, Paraprevotella.clara
## 2024-12-18 19:17:24.784197 INFO::Fitting model to feature number 31, Paraprevotella.unclassified
## 2024-12-18 19:17:24.926888 INFO::Fitting model to feature number 32, Prevotella.copri
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:25.065999 INFO::Fitting model to feature number 33, Alistipes.finegoldii
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:25.191013 INFO::Fitting model to feature number 34, Alistipes.onderdonkii
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:25.319435 INFO::Fitting model to feature number 35, Alistipes.putredinis
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:25.462472 INFO::Fitting model to feature number 36, Alistipes.shahii
## 2024-12-18 19:17:25.594163 INFO::Fitting model to feature number 37, Alistipes.unclassified
## 2024-12-18 19:17:25.719517 INFO::Fitting model to feature number 38, Streptococcus.salivarius
## 2024-12-18 19:17:25.886699 INFO::Fitting model to feature number 39, Clostridium.bolteae
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:26.092466 INFO::Fitting model to feature number 40, Clostridium.citroniae
## boundary (singular) fit: see help('isSingular')
## Feature Clostridium.citroniae : simpleWarning: Model failed to converge with 1 negative eigenvalue: -3.4e+01
## 2024-12-18 19:17:26.249314 WARNING::Fitting problem for feature 40 a warning was issued
## boundary (singular) fit: see help('isSingular')
## Warning: Model failed to converge with 1 negative eigenvalue: -3.4e+01
## 2024-12-18 19:17:26.45175 INFO::Fitting model to feature number 41, Clostridium.clostridioforme
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:26.628395 INFO::Fitting model to feature number 42, Clostridium.hathewayi
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:26.806394 INFO::Fitting model to feature number 43, Clostridium.leptum
## 2024-12-18 19:17:26.998336 INFO::Fitting model to feature number 44, Clostridium.nexile
## 2024-12-18 19:17:27.128258 INFO::Fitting model to feature number 45, Clostridium.symbiosum
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:27.253939 INFO::Fitting model to feature number 46, Flavonifractor.plautii
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:27.390723 INFO::Fitting model to feature number 47, Eubacterium.eligens
## 2024-12-18 19:17:27.517579 INFO::Fitting model to feature number 48, Eubacterium.hallii
## 2024-12-18 19:17:27.649366 INFO::Fitting model to feature number 49, Eubacterium.rectale
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:27.799294 INFO::Fitting model to feature number 50, Eubacterium.siraeum
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:27.935324 INFO::Fitting model to feature number 51, Eubacterium.sp.3.1.31
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:28.096659 INFO::Fitting model to feature number 52, Eubacterium.ventriosum
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:28.220925 INFO::Fitting model to feature number 53, Ruminococcus.gnavus
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:28.358259 INFO::Fitting model to feature number 54, Ruminococcus.obeum
## 2024-12-18 19:17:28.490912 INFO::Fitting model to feature number 55, Ruminococcus.torques
## 2024-12-18 19:17:28.66802 INFO::Fitting model to feature number 56, Coprococcus.comes
## 2024-12-18 19:17:28.852936 INFO::Fitting model to feature number 57, Dorea.longicatena
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:29.005338 INFO::Fitting model to feature number 58, Lachnospiraceae.bacterium.1.1.57FAA
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:29.145481 INFO::Fitting model to feature number 59, Lachnospiraceae.bacterium.3.1.46FAA
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:29.269976 INFO::Fitting model to feature number 60, Roseburia.hominis
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:29.422398 INFO::Fitting model to feature number 61, Roseburia.intestinalis
## 2024-12-18 19:17:29.539752 INFO::Fitting model to feature number 62, Roseburia.inulinivorans
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:29.668075 INFO::Fitting model to feature number 63, Roseburia.unclassified
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:29.789991 INFO::Fitting model to feature number 64, Oscillibacter.unclassified
## 2024-12-18 19:17:29.9335 INFO::Fitting model to feature number 65, Peptostreptococcaceae.noname.unclassified
## 2024-12-18 19:17:30.059675 INFO::Fitting model to feature number 66, Faecalibacterium.prausnitzii
## 2024-12-18 19:17:30.195149 INFO::Fitting model to feature number 67, Ruminococcus.bromii
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:30.316012 INFO::Fitting model to feature number 68, Ruminococcus.callidus
## 2024-12-18 19:17:30.712378 INFO::Fitting model to feature number 69, Ruminococcus.lactaris
## 2024-12-18 19:17:30.879885 INFO::Fitting model to feature number 70, Subdoligranulum.unclassified
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:31.067253 INFO::Fitting model to feature number 71, Coprobacillus.unclassified
## 2024-12-18 19:17:31.246088 INFO::Fitting model to feature number 72, Acidaminococcus.unclassified
## 2024-12-18 19:17:31.401501 INFO::Fitting model to feature number 73, Dialister.invisus
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:31.571226 INFO::Fitting model to feature number 74, Veillonella.atypica
## 2024-12-18 19:17:31.714306 INFO::Fitting model to feature number 75, Veillonella.dispar
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:31.857013 INFO::Fitting model to feature number 76, Veillonella.parvula
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:31.98057 INFO::Fitting model to feature number 77, Veillonella.unclassified
## 2024-12-18 19:17:32.111166 INFO::Fitting model to feature number 78, Burkholderiales.bacterium.1.1.47
## 2024-12-18 19:17:32.246906 INFO::Fitting model to feature number 79, Parasutterella.excrementihominis
## 2024-12-18 19:17:32.38321 INFO::Fitting model to feature number 80, Sutterella.wadsworthensis
## 2024-12-18 19:17:32.533185 INFO::Fitting model to feature number 81, Bilophila.unclassified
## 2024-12-18 19:17:32.668633 INFO::Fitting model to feature number 82, Escherichia.coli
## 2024-12-18 19:17:32.79355 INFO::Fitting model to feature number 83, Escherichia.unclassified
## 2024-12-18 19:17:32.96273 INFO::Fitting model to feature number 84, Klebsiella.pneumoniae
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:33.154902 INFO::Fitting model to feature number 85, Haemophilus.parainfluenzae
## boundary (singular) fit: see help('isSingular')
## 2024-12-18 19:17:33.348285 INFO::Fitting model to feature number 86, Akkermansia.muciniphila
## 2024-12-18 19:17:33.499318 INFO::Fitting model to feature number 87, C2likevirus.unclassified
## 2024-12-18 19:17:33.752324 INFO::Counting total values for each feature
## 2024-12-18 19:17:33.816858 INFO::Writing filtered data to file demo_output/features/filtered_data.tsv
## 2024-12-18 19:17:34.139603 INFO::Writing filtered, normalized data to file demo_output/features/filtered_data_norm.tsv
## 2024-12-18 19:17:34.539091 INFO::Writing filtered, normalized, transformed data to file demo_output/features/filtered_data_norm_transformed.tsv
## 2024-12-18 19:17:35.022408 INFO::Writing residuals to file demo_output/fits/residuals.rds
## 2024-12-18 19:17:35.086004 INFO::Writing fitted values to file demo_output/fits/fitted.rds
## 2024-12-18 19:17:35.122627 INFO::Writing extracted random effects to file demo_output/fits/ranef.rds
## 2024-12-18 19:17:35.129302 INFO::Writing all results to file (ordered by increasing q-values): demo_output/all_results.tsv
## 2024-12-18 19:17:35.14702 INFO::Writing the significant results (those which are less than or equal to the threshold of 0.250000 ) to file (ordered by increasing q-values): demo_output/significant_results.tsv
## 2024-12-18 19:17:35.158717 INFO::Writing heatmap of significant results to file: demo_output/heatmap.pdf
## 2024-12-18 19:17:35.444699 INFO::Writing association plots (one for each significant association) to output folder: demo_output
## 2024-12-18 19:17:35.45193 INFO::Plotting associations from most to least significant, grouped by metadata
## 2024-12-18 19:17:35.45375 INFO::Plotting data for metadata number 1, dysbiosisCD
## 2024-12-18 19:17:35.456297 INFO::Creating boxplot for categorical data, dysbiosisCD vs Faecalibacterium.prausnitzii
## 2024-12-18 19:17:35.882332 INFO::Creating boxplot for categorical data, dysbiosisCD vs Subdoligranulum.unclassified
## 2024-12-18 19:17:36.201174 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.uniformis
## 2024-12-18 19:17:36.539806 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.rectale
## 2024-12-18 19:17:36.886899 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.vulgatus
## 2024-12-18 19:17:37.199407 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.ovatus
## 2024-12-18 19:17:37.454979 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.obeum
## 2024-12-18 19:17:37.69773 INFO::Creating boxplot for categorical data, dysbiosisCD vs Oscillibacter.unclassified
## 2024-12-18 19:17:37.956921 INFO::Creating boxplot for categorical data, dysbiosisCD vs Roseburia.inulinivorans
## 2024-12-18 19:17:38.20715 INFO::Creating boxplot for categorical data, dysbiosisCD vs Roseburia.hominis
## 2024-12-18 19:17:38.565523 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.thetaiotaomicron
## 2024-12-18 19:17:38.918322 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.putredinis
## 2024-12-18 19:17:39.175701 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.distasonis
## 2024-12-18 19:17:39.438252 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.dorei
## 2024-12-18 19:17:39.813644 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.shahii
## 2024-12-18 19:17:40.077059 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.xylanisolvens
## 2024-12-18 19:17:40.358297 INFO::Creating boxplot for categorical data, dysbiosisCD vs Clostridium.leptum
## 2024-12-18 19:17:40.699538 INFO::Creating boxplot for categorical data, dysbiosisCD vs Dorea.longicatena
## 2024-12-18 19:17:41.072126 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.caccae
## 2024-12-18 19:17:41.455668 INFO::Creating boxplot for categorical data, dysbiosisCD vs Lachnospiraceae.bacterium.3.1.46FAA
## 2024-12-18 19:17:41.854037 INFO::Creating boxplot for categorical data, dysbiosisCD vs Escherichia.coli
## 2024-12-18 19:17:42.201786 INFO::Creating boxplot for categorical data, dysbiosisCD vs Klebsiella.pneumoniae
## 2024-12-18 19:17:42.593478 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bilophila.unclassified
## 2024-12-18 19:17:42.969141 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.finegoldii
## 2024-12-18 19:17:43.237996 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.eligens
## 2024-12-18 19:17:43.485688 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.stercoris
## 2024-12-18 19:17:43.847289 INFO::Creating boxplot for categorical data, dysbiosisCD vs Coprococcus.comes
## 2024-12-18 19:17:44.093181 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.fragilis
## 2024-12-18 19:17:44.373442 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.finegoldii
## 2024-12-18 19:17:44.614326 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.hallii
## 2024-12-18 19:17:44.874265 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.faecis
## 2024-12-18 19:17:45.141494 INFO::Creating boxplot for categorical data, dysbiosisCD vs Paraprevotella.clara
## 2024-12-18 19:17:45.537566 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.siraeum
## 2024-12-18 19:17:46.084896 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.merdae
## 2024-12-18 19:17:46.433171 INFO::Creating boxplot for categorical data, dysbiosisCD vs Paraprevotella.unclassified
## 2024-12-18 19:17:46.682293 INFO::Creating boxplot for categorical data, dysbiosisCD vs Collinsella.aerofaciens
## 2024-12-18 19:17:46.950967 INFO::Creating boxplot for categorical data, dysbiosisCD vs Odoribacter.splanchnicus
## 2024-12-18 19:17:47.201684 INFO::Creating boxplot for categorical data, dysbiosisCD vs Clostridium.clostridioforme
## 2024-12-18 19:17:47.461948 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.eggerthii
## 2024-12-18 19:17:47.721066 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.onderdonkii
## 2024-12-18 19:17:48.076876 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.ventriosum
## 2024-12-18 19:17:48.480962 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.lactaris
## 2024-12-18 19:17:48.859664 INFO::Creating boxplot for categorical data, dysbiosisCD vs Burkholderiales.bacterium.1.1.47
## 2024-12-18 19:17:49.111781 INFO::Creating boxplot for categorical data, dysbiosisCD vs Dialister.invisus
## 2024-12-18 19:17:49.423639 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.bromii
## 2024-12-18 19:17:49.699662 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parasutterella.excrementihominis
## 2024-12-18 19:17:50.013935 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.unclassified
## 2024-12-18 19:17:50.355013 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.torques
## 2024-12-18 19:17:50.722188 INFO::Creating boxplot for categorical data, dysbiosisCD vs Coprobacillus.unclassified
## 2024-12-18 19:17:50.972541 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.cellulosilyticus
## 2024-12-18 19:17:51.286446 INFO::Creating boxplot for categorical data, dysbiosisCD vs Roseburia.intestinalis
## 2024-12-18 19:17:51.644543 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.unclassified
## 2024-12-18 19:17:51.997527 INFO::Creating boxplot for categorical data, dysbiosisCD vs Acidaminococcus.unclassified
## 2024-12-18 19:17:52.358688 INFO::Creating boxplot for categorical data, dysbiosisCD vs Barnesiella.intestinihominis
## 2024-12-18 19:17:52.737895 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.massiliensis
## 2024-12-18 19:17:53.096879 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.sp.3.1.31
## 2024-12-18 19:17:53.500456 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.salyersiae
## 2024-12-18 19:17:53.79013 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroidales.bacterium.ph8
## 2024-12-18 19:17:54.151245 INFO::Creating boxplot for categorical data, dysbiosisCD vs Clostridium.citroniae
## 2024-12-18 19:17:54.427327 INFO::Creating boxplot for categorical data, dysbiosisCD vs Flavonifractor.plautii
## 2024-12-18 19:17:54.771101 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.goldsteinii
## 2024-12-18 19:17:55.16489 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bifidobacterium.longum
## 2024-12-18 19:17:55.567198 INFO::Creating boxplot for categorical data, dysbiosisCD vs Lachnospiraceae.bacterium.1.1.57FAA
## 2024-12-18 19:17:59.587088 INFO::Plotting data for metadata number 2, dysbiosisUC
## 2024-12-18 19:17:59.590032 INFO::Creating boxplot for categorical data, dysbiosisUC vs Subdoligranulum.unclassified
## 2024-12-18 19:17:59.874654 INFO::Creating boxplot for categorical data, dysbiosisUC vs Faecalibacterium.prausnitzii
## 2024-12-18 19:18:00.169843 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.caccae
## 2024-12-18 19:18:00.508677 INFO::Creating boxplot for categorical data, dysbiosisUC vs Oscillibacter.unclassified
## 2024-12-18 19:18:00.766221 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.uniformis
## 2024-12-18 19:18:01.083648 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.siraeum
## 2024-12-18 19:18:01.396915 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.ovatus
## 2024-12-18 19:18:01.740551 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.fragilis
## 2024-12-18 19:18:02.060622 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.shahii
## 2024-12-18 19:18:02.322744 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.rectale
## 2024-12-18 19:18:02.5735 INFO::Creating boxplot for categorical data, dysbiosisUC vs Roseburia.hominis
## 2024-12-18 19:18:03.221923 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.putredinis
## 2024-12-18 19:18:03.56093 INFO::Creating boxplot for categorical data, dysbiosisUC vs Lachnospiraceae.bacterium.3.1.46FAA
## 2024-12-18 19:18:03.877784 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.hallii
## 2024-12-18 19:18:04.136104 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.xylanisolvens
## 2024-12-18 19:18:04.404434 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.stercoris
## 2024-12-18 19:18:04.66315 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.finegoldii
## 2024-12-18 19:18:04.964876 INFO::Creating boxplot for categorical data, dysbiosisUC vs Barnesiella.intestinihominis
## 2024-12-18 19:18:05.234865 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.leptum
## 2024-12-18 19:18:05.50786 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.gnavus
## 2024-12-18 19:18:05.796606 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.onderdonkii
## 2024-12-18 19:18:06.055273 INFO::Creating boxplot for categorical data, dysbiosisUC vs Flavonifractor.plautii
## 2024-12-18 19:18:06.34202 INFO::Creating boxplot for categorical data, dysbiosisUC vs Parabacteroides.merdae
## 2024-12-18 19:18:06.617592 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.cellulosilyticus
## 2024-12-18 19:18:06.889529 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bifidobacterium.longum
## 2024-12-18 19:18:07.154251 INFO::Creating boxplot for categorical data, dysbiosisUC vs Klebsiella.pneumoniae
## 2024-12-18 19:18:07.41103 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.citroniae
## 2024-12-18 19:18:07.665643 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.ventriosum
## 2024-12-18 19:18:07.964981 INFO::Creating boxplot for categorical data, dysbiosisUC vs Parabacteroides.distasonis
## 2024-12-18 19:18:08.244403 INFO::Creating boxplot for categorical data, dysbiosisUC vs Parabacteroides.goldsteinii
## 2024-12-18 19:18:08.501303 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.torques
## 2024-12-18 19:18:08.737172 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroidales.bacterium.ph8
## 2024-12-18 19:18:09.055278 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.obeum
## 2024-12-18 19:18:09.293571 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bifidobacterium.bifidum
## 2024-12-18 19:18:09.558737 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bifidobacterium.adolescentis
## 2024-12-18 19:18:09.79149 INFO::Creating boxplot for categorical data, dysbiosisUC vs Collinsella.aerofaciens
## 2024-12-18 19:18:10.093696 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.hathewayi
## 2024-12-18 19:18:10.344646 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bilophila.unclassified
## 2024-12-18 19:18:10.613996 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.eligens
## 2024-12-18 19:18:10.871128 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.vulgatus
## 2024-12-18 19:18:11.165562 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.bolteae
## 2024-12-18 19:18:11.42231 INFO::Creating boxplot for categorical data, dysbiosisUC vs Dialister.invisus
## 2024-12-18 19:18:11.745701 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.lactaris
## 2024-12-18 19:18:12.097565 INFO::Creating boxplot for categorical data, dysbiosisUC vs Burkholderiales.bacterium.1.1.47
## 2024-12-18 19:18:16.667311 INFO::Plotting data for metadata number 3, dysbiosisnonIBD
## 2024-12-18 19:18:16.671426 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Faecalibacterium.prausnitzii
## 2024-12-18 19:18:16.999834 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.rectale
## 2024-12-18 19:18:17.300594 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.sp.3.1.31
## 2024-12-18 19:18:17.642143 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.hominis
## 2024-12-18 19:18:17.986831 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Subdoligranulum.unclassified
## 2024-12-18 19:18:18.363762 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Lachnospiraceae.bacterium.3.1.46FAA
## 2024-12-18 19:18:18.699544 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Ruminococcus.torques
## 2024-12-18 19:18:19.070503 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Clostridium.leptum
## 2024-12-18 19:18:19.334764 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Odoribacter.splanchnicus
## 2024-12-18 19:18:19.72645 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Escherichia.coli
## 2024-12-18 19:18:20.037292 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Ruminococcus.obeum
## 2024-12-18 19:18:20.434706 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bilophila.unclassified
## 2024-12-18 19:18:20.799531 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.uniformis
## 2024-12-18 19:18:21.08306 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.eligens
## 2024-12-18 19:18:21.493241 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Klebsiella.pneumoniae
## 2024-12-18 19:18:21.899013 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Ruminococcus.bromii
## 2024-12-18 19:18:22.312071 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.vulgatus
## 2024-12-18 19:18:22.574114 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs C2likevirus.unclassified
## 2024-12-18 19:18:22.862205 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bifidobacterium.adolescentis
## 2024-12-18 19:18:23.1066 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Alistipes.finegoldii
## 2024-12-18 19:18:23.369126 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.inulinivorans
## 2024-12-18 19:18:23.747438 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Alistipes.onderdonkii
## 2024-12-18 19:18:24.173856 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.unclassified
## 2024-12-18 19:18:24.471308 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.hallii
## 2024-12-18 19:18:25.075021 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.intestinalis
## 2024-12-18 19:18:25.358342 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Prevotella.copri
## 2024-12-18 19:18:25.61795 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.dorei
## 2024-12-18 19:18:26.202225 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.fragilis
## 2024-12-18 19:18:26.558278 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Alistipes.shahii
## 2024-12-18 19:18:30.011456 INFO::Plotting data for metadata number 4, antibiotics
## 2024-12-18 19:18:30.014199 INFO::Creating boxplot for categorical data, antibiotics vs Roseburia.inulinivorans
## 2024-12-18 19:18:30.228499 INFO::Creating boxplot for categorical data, antibiotics vs Roseburia.hominis
## 2024-12-18 19:18:30.450104 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.rectale
## 2024-12-18 19:18:30.67598 INFO::Creating boxplot for categorical data, antibiotics vs Dialister.invisus
## 2024-12-18 19:18:30.906727 INFO::Creating boxplot for categorical data, antibiotics vs Roseburia.intestinalis
## 2024-12-18 19:18:31.125835 INFO::Creating boxplot for categorical data, antibiotics vs Dorea.longicatena
## 2024-12-18 19:18:31.365395 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.callidus
## 2024-12-18 19:18:31.591693 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.bromii
## 2024-12-18 19:18:31.819485 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.obeum
## 2024-12-18 19:18:32.038595 INFO::Creating boxplot for categorical data, antibiotics vs Klebsiella.pneumoniae
## 2024-12-18 19:18:32.269578 INFO::Creating boxplot for categorical data, antibiotics vs Bifidobacterium.adolescentis
## 2024-12-18 19:18:32.476657 INFO::Creating boxplot for categorical data, antibiotics vs Faecalibacterium.prausnitzii
## 2024-12-18 19:18:32.698634 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.hallii
## 2024-12-18 19:18:32.927111 INFO::Creating boxplot for categorical data, antibiotics vs Bilophila.unclassified
## 2024-12-18 19:18:33.14621 INFO::Creating boxplot for categorical data, antibiotics vs Clostridium.leptum
## 2024-12-18 19:18:33.372101 INFO::Creating boxplot for categorical data, antibiotics vs Lachnospiraceae.bacterium.3.1.46FAA
## 2024-12-18 19:18:33.6074 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.finegoldii
## 2024-12-18 19:18:33.89803 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.sp.3.1.31
## 2024-12-18 19:18:34.117006 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.fragilis
## 2024-12-18 19:18:34.341487 INFO::Creating boxplot for categorical data, antibiotics vs Alistipes.onderdonkii
## 2024-12-18 19:18:34.566776 INFO::Creating boxplot for categorical data, antibiotics vs Sutterella.wadsworthensis
## 2024-12-18 19:18:34.796916 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.eligens
## 2024-12-18 19:18:35.026972 INFO::Creating boxplot for categorical data, antibiotics vs Collinsella.aerofaciens
## 2024-12-18 19:18:35.301191 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.thetaiotaomicron
## 2024-12-18 19:18:35.547931 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.eggerthii
## 2024-12-18 19:18:35.798605 INFO::Creating boxplot for categorical data, antibiotics vs Haemophilus.parainfluenzae
## 2024-12-18 19:18:36.082735 INFO::Creating boxplot for categorical data, antibiotics vs Bifidobacterium.pseudocatenulatum
## 2024-12-18 19:18:36.398539 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.torques
## 2024-12-18 19:18:36.667423 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.ventriosum
## 2024-12-18 19:18:36.916077 INFO::Creating boxplot for categorical data, antibiotics vs Parasutterella.excrementihominis
## 2024-12-18 19:18:37.146576 INFO::Creating boxplot for categorical data, antibiotics vs Peptostreptococcaceae.noname.unclassified
## 2024-12-18 19:18:37.391837 INFO::Creating boxplot for categorical data, antibiotics vs Veillonella.dispar
## 2024-12-18 19:18:37.620725 INFO::Creating boxplot for categorical data, antibiotics vs Veillonella.atypica
## 2024-12-18 19:18:37.857107 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroidales.bacterium.ph8
## 2024-12-18 19:18:38.083225 INFO::Creating boxplot for categorical data, antibiotics vs Clostridium.nexile
## 2024-12-18 19:18:38.38941 INFO::Creating boxplot for categorical data, antibiotics vs Burkholderiales.bacterium.1.1.47
## 2024-12-18 19:18:38.609388 INFO::Creating boxplot for categorical data, antibiotics vs Lachnospiraceae.bacterium.1.1.57FAA
## 2024-12-18 19:18:38.85703 INFO::Creating boxplot for categorical data, antibiotics vs Akkermansia.muciniphila
## 2024-12-18 19:18:39.083647 INFO::Creating boxplot for categorical data, antibiotics vs Clostridium.citroniae
## 2024-12-18 19:18:39.344906 INFO::Creating boxplot for categorical data, antibiotics vs Odoribacter.splanchnicus
## 2024-12-18 19:18:42.860685 INFO::Plotting data for metadata number 5, age
## 2024-12-18 19:18:42.863542 INFO::Creating scatter plot for continuous data, age vs Haemophilus.parainfluenzae
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:43.180245 INFO::Creating scatter plot for continuous data, age vs Bifidobacterium.pseudocatenulatum
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:43.581232 INFO::Creating scatter plot for continuous data, age vs Faecalibacterium.prausnitzii
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:43.970129 INFO::Creating scatter plot for continuous data, age vs Clostridium.clostridioforme
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:44.340131 INFO::Creating scatter plot for continuous data, age vs Veillonella.parvula
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:44.620048 INFO::Creating scatter plot for continuous data, age vs Subdoligranulum.unclassified
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:44.987742 INFO::Creating scatter plot for continuous data, age vs Clostridium.symbiosum
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:45.385215 INFO::Creating scatter plot for continuous data, age vs Ruminococcus.gnavus
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:45.758712 INFO::Creating scatter plot for continuous data, age vs Dialister.invisus
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:46.034596 INFO::Creating scatter plot for continuous data, age vs Veillonella.dispar
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:46.319864 INFO::Creating scatter plot for continuous data, age vs Veillonella.unclassified
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:46.581899 INFO::Creating scatter plot for continuous data, age vs Bacteroides.thetaiotaomicron
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:46.897846 INFO::Creating scatter plot for continuous data, age vs Ruminococcus.bromii
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:47.22364 INFO::Creating scatter plot for continuous data, age vs Bacteroides.intestinalis
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:47.504864 INFO::Creating scatter plot for continuous data, age vs Eubacterium.siraeum
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:47.804382 INFO::Creating scatter plot for continuous data, age vs Prevotella.copri
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:48.105739 INFO::Creating scatter plot for continuous data, age vs Alistipes.unclassified
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:48.341561 INFO::Creating scatter plot for continuous data, age vs Bacteroidales.bacterium.ph8
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:48.585129 INFO::Creating scatter plot for continuous data, age vs Bifidobacterium.longum
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:48.810612 INFO::Creating scatter plot for continuous data, age vs Akkermansia.muciniphila
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:49.145037 INFO::Creating scatter plot for continuous data, age vs Collinsella.aerofaciens
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:49.565448 INFO::Creating scatter plot for continuous data, age vs Parabacteroides.distasonis
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values or values outside the scale range
## (`geom_point()`).
## 2024-12-18 19:18:54.336949 INFO::Plotting data for metadata number 6, diagnosis
## 2024-12-18 19:18:54.341135 INFO::Creating boxplot for categorical data, diagnosis vs Bifidobacterium.adolescentis
## 2024-12-18 19:18:54.683395 INFO::Creating boxplot for categorical data, diagnosis vs Akkermansia.muciniphila
## 2024-12-18 19:18:55.029672 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.bolteae
## 2024-12-18 19:18:55.339993 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.putredinis
## 2024-12-18 19:18:55.645927 INFO::Creating boxplot for categorical data, diagnosis vs Coprobacillus.unclassified
## 2024-12-18 19:18:56.082863 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.clostridioforme
## 2024-12-18 19:18:56.477269 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.shahii
## 2024-12-18 19:18:56.893993 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.bromii
## 2024-12-18 19:18:57.247242 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.leptum
## 2024-12-18 19:18:57.549784 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.symbiosum
## 2024-12-18 19:18:57.822028 INFO::Creating boxplot for categorical data, diagnosis vs Roseburia.inulinivorans
## 2024-12-18 19:18:58.206203 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.bromii
## 2024-12-18 19:18:58.555113 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.shahii
## 2024-12-18 19:18:58.906102 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.finegoldii
## 2024-12-18 19:18:59.230773 INFO::Creating boxplot for categorical data, diagnosis vs Parabacteroides.goldsteinii
## 2024-12-18 19:18:59.480123 INFO::Creating boxplot for categorical data, diagnosis vs Bilophila.unclassified
## 2024-12-18 19:18:59.740644 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.leptum
## 2024-12-18 19:19:00.052708 INFO::Creating boxplot for categorical data, diagnosis vs Roseburia.hominis
## 2024-12-18 19:19:00.321888 INFO::Creating boxplot for categorical data, diagnosis vs Coprobacillus.unclassified
## 2024-12-18 19:19:00.56628 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.onderdonkii
## 2024-12-18 19:19:00.825706 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.ventriosum
## 2024-12-18 19:19:01.221652 INFO::Creating boxplot for categorical data, diagnosis vs Roseburia.hominis
## 2024-12-18 19:19:01.596133 INFO::Creating boxplot for categorical data, diagnosis vs Sutterella.wadsworthensis
## 2024-12-18 19:19:01.913939 INFO::Creating boxplot for categorical data, diagnosis vs Akkermansia.muciniphila
## 2024-12-18 19:19:02.34987 INFO::Creating boxplot for categorical data, diagnosis vs Sutterella.wadsworthensis
## 2024-12-18 19:19:02.736417 INFO::Creating boxplot for categorical data, diagnosis vs Subdoligranulum.unclassified
## 2024-12-18 19:19:02.993727 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.symbiosum
## 2024-12-18 19:19:03.387545 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.onderdonkii
## 2024-12-18 19:19:03.685405 INFO::Creating boxplot for categorical data, diagnosis vs Parabacteroides.unclassified
## 2024-12-18 19:19:04.064726 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.lactaris
## 2024-12-18 19:19:04.318022 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.callidus
## 2024-12-18 19:19:04.584814 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.gnavus
## 2024-12-18 19:19:04.875877 INFO::Creating boxplot for categorical data, diagnosis vs Bacteroides.fragilis
## 2024-12-18 19:19:05.143483 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.rectale
## 2024-12-18 19:19:05.505928 INFO::Creating boxplot for categorical data, diagnosis vs Odoribacter.splanchnicus
## 2024-12-18 19:19:05.810628 INFO::Creating boxplot for categorical data, diagnosis vs Parabacteroides.distasonis
## 2024-12-18 19:19:06.214125 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.rectale
## 2024-12-18 19:19:06.623661 INFO::Creating boxplot for categorical data, diagnosis vs Bacteroides.finegoldii
## 2024-12-18 19:19:07.069183 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.callidus
## 2024-12-18 19:19:07.351871 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.finegoldii
## 2024-12-18 19:19:07.632011 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.putredinis
## 2024-12-18 19:19:07.897606 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.bolteae
## 2024-12-18 19:19:08.275397 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.siraeum
Session Info

Session info from running the demo in R can be displayed with the following command.

sessionInfo()
## R Under development (unstable) (2024-10-26 r87273 ucrt)
## Platform: x86_64-w64-mingw32/x64
## Running under: Windows Server 2022 x64 (build 20348)
## 
## Matrix products: default
## 
## 
## locale:
## [1] LC_COLLATE=C                          
## [2] LC_CTYPE=English_United States.utf8   
## [3] LC_MONETARY=English_United States.utf8
## [4] LC_NUMERIC=C                          
## [5] LC_TIME=English_United States.utf8    
## 
## time zone: America/New_York
## tzcode source: internal
## 
## attached base packages:
## [1] stats     graphics  grDevices utils     datasets  methods   base     
## 
## other attached packages:
## [1] Maaslin2_1.21.0
## 
## loaded via a namespace (and not attached):
##  [1] sass_0.4.9          generics_0.1.3      robustbase_0.99-4-1
##  [4] lattice_0.22-6      lme4_1.1-35.5       digest_0.6.37      
##  [7] magrittr_2.0.3      RColorBrewer_1.1-3  evaluate_1.0.1     
## [10] grid_4.5.0          mvtnorm_1.3-2       fastmap_1.2.0      
## [13] jsonlite_1.8.9      Matrix_1.7-1        DBI_1.2.3          
## [16] optparse_1.7.5      biglm_0.9-3         mgcv_1.9-1         
## [19] scales_1.3.0        pbapply_1.7-2       permute_0.9-7      
## [22] numDeriv_2016.8-1.1 getopt_1.20.4       jquerylib_0.1.4    
## [25] cli_3.6.3           rlang_1.1.4         crayon_1.5.3       
## [28] munsell_0.5.1       splines_4.5.0       withr_3.0.2        
## [31] cachem_1.1.0        yaml_2.3.10         vegan_2.6-8        
## [34] tools_4.5.0         parallel_4.5.0      nloptr_2.1.1       
## [37] minqa_1.2.8         dplyr_1.1.4         colorspace_2.1-1   
## [40] ggplot2_3.5.1       boot_1.3-31         hash_2.2.6.3       
## [43] vctrs_0.6.5         R6_2.5.1            lifecycle_1.0.4    
## [46] MASS_7.3-61         logging_0.10-108    pcaPP_2.0-5        
## [49] cluster_2.1.8       pkgconfig_2.0.3     gtable_0.3.6       
## [52] lmerTest_3.1-3      pillar_1.10.0       bslib_0.8.0        
## [55] Rcpp_1.0.13-1       data.table_1.16.4   glue_1.8.0         
## [58] DEoptimR_1.1-3-1    xfun_0.49           tibble_3.2.1       
## [61] tidyselect_1.2.1    knitr_1.49          farver_2.1.2       
## [64] htmltools_0.5.8.1   nlme_3.1-166        labeling_0.4.3     
## [67] rmarkdown_2.29      pheatmap_1.0.12     compiler_4.5.0

Options

Run MaAsLin2 help to print a list of the options and the default settings.

$ Maaslin2.R –help Usage: ./R/Maaslin2.R options <data.tsv> <metadata.tsv>

Options: -h, –help Show this help message and exit

-a MIN_ABUNDANCE, --min_abundance=MIN_ABUNDANCE
    The minimum abundance for each feature [ Default: 0 ]

-p MIN_PREVALENCE, --min_prevalence=MIN_PREVALENCE
    The minimum percent of samples for which a feature 
    is detected at minimum abundance [ Default: 0.1 ]

-b MIN_VARIANCE, --min_variance=MIN_VARIANCE
    Keep features with variance greater than [ Default: 0.0 ]

-s MAX_SIGNIFICANCE, --max_significance=MAX_SIGNIFICANCE
    The q-value threshold for significance [ Default: 0.25 ]

-n NORMALIZATION, --normalization=NORMALIZATION
    The normalization method to apply [ Default: TSS ]
    [ Choices: TSS, CLR, CSS, NONE, TMM ]

-t TRANSFORM, --transform=TRANSFORM
    The transform to apply [ Default: LOG ]
    [ Choices: LOG, LOGIT, AST, NONE ]

-m ANALYSIS_METHOD, --analysis_method=ANALYSIS_METHOD
    The analysis method to apply [ Default: LM ]
    [ Choices: LM, CPLM, NEGBIN, ZINB ]

-r RANDOM_EFFECTS, --random_effects=RANDOM_EFFECTS
    The random effects for the model, comma-delimited
    for multiple effects [ Default: none ]

-f FIXED_EFFECTS, --fixed_effects=FIXED_EFFECTS
    The fixed effects for the model, comma-delimited
    for multiple effects [ Default: all ]

-c CORRECTION, --correction=CORRECTION
    The correction method for computing the 
    q-value [ Default: BH ]

-z STANDARDIZE, --standardize=STANDARDIZE
    Apply z-score so continuous metadata are 
    on the same scale [ Default: TRUE ]

-l PLOT_HEATMAP, --plot_heatmap=PLOT_HEATMAP
    Generate a heatmap for the significant 
    associations [ Default: TRUE ]

-i HEATMAP_FIRST_N, --heatmap_first_n=HEATMAP_FIRST_N
    In heatmap, plot top N features with significant 
    associations [ Default: TRUE ]

-o PLOT_SCATTER, --plot_scatter=PLOT_SCATTER
    Generate scatter plots for the significant
    associations [ Default: TRUE ]
    
-g MAX_PNGS, --max_pngs=MAX_PNGS
    The maximum number of scatter plots for signficant associations 
    to save as png files [ Default: 10 ]

-O SAVE_SCATTER, --save_scatter=SAVE_SCATTER
    Save all scatter plot ggplot objects
    to an RData file [ Default: FALSE ]

-e CORES, --cores=CORES
    The number of R processes to run in parallel
    [ Default: 1 ]
    
-j SAVE_MODELS --save_models=SAVE_MODELS
    Return the full model outputs and save to an RData file
    [ Default: FALSE ]

-d REFERENCE, --reference=REFERENCE
    The factor to use as a reference level for a categorical variable 
    provided as a string of 'variable,reference', semi-colon delimited for 
    multiple variables. Not required if metadata is passed as a factor or 
    for variables with less than two levels but can be set regardless.
    [ Default: NA ] 

Troubleshooting

  1. Question: When I run from the command line I see the error Maaslin2.R: command not found. How do I fix this?
    • Answer: Provide the full path to the executable when running Maaslin2.R.
  2. Question: When I run as a function I see the error Error in library(Maaslin2): there is no package called 'Maaslin2'. How do I fix this?
    • Answer: Install the R package and then try loading the library again.
  3. Question: When I try to install the R package I see errors about dependencies not being installed. Why is this?
    • Answer: Installing the R package will not automatically install the packages MaAsLin2 requires. Please install the dependencies and then install the MaAsLin2 R package.