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"
## 2024-04-17 18:36:05.077446 INFO::Writing function arguments to log file
## 2024-04-17 18:36:05.124365 INFO::Verifying options selected are valid
## 2024-04-17 18:36:05.177695 INFO::Determining format of input files
## 2024-04-17 18:36:05.181171 INFO::Input format is data samples as rows and metadata samples as rows
## 2024-04-17 18:36:05.193301 INFO::Formula for random effects: expr ~ (1 | site) + (1 | subject)
## 2024-04-17 18:36:05.19675 INFO::Formula for fixed effects: expr ~  diagnosis + dysbiosisnonIBD + dysbiosisUC + dysbiosisCD + antibiotics + age
## 2024-04-17 18:36:05.202061 INFO::Filter data based on min abundance and min prevalence
## 2024-04-17 18:36:05.204671 INFO::Total samples in data: 1595
## 2024-04-17 18:36:05.207078 INFO::Min samples required with min abundance for a feature not to be filtered: 159.500000
## 2024-04-17 18:36:05.214944 INFO::Total filtered features: 0
## 2024-04-17 18:36:05.218087 INFO::Filtered feature names from abundance and prevalence filtering:
## 2024-04-17 18:36:05.232887 INFO::Total filtered features with variance filtering: 0
## 2024-04-17 18:36:05.235844 INFO::Filtered feature names from variance filtering:
## 2024-04-17 18:36:05.238578 INFO::Running selected normalization method: TSS
## 2024-04-17 18:36:06.471564 INFO::Bypass z-score application to metadata
## 2024-04-17 18:36:06.473527 INFO::Running selected transform method: LOG
## 2024-04-17 18:36:06.49732 INFO::Running selected analysis method: LM
## 2024-04-17 18:36:06.78757 INFO::Fitting model to feature number 1, Bifidobacterium.adolescentis
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:07.23453 INFO::Fitting model to feature number 2, Bifidobacterium.bifidum
## 2024-04-17 18:36:07.620896 INFO::Fitting model to feature number 3, Bifidobacterium.longum
## 2024-04-17 18:36:07.784947 INFO::Fitting model to feature number 4, Bifidobacterium.pseudocatenulatum
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:07.958674 INFO::Fitting model to feature number 5, Collinsella.aerofaciens
## 2024-04-17 18:36:08.203346 INFO::Fitting model to feature number 6, Bacteroides.caccae
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:08.443889 INFO::Fitting model to feature number 7, Bacteroides.cellulosilyticus
## 2024-04-17 18:36:08.681001 INFO::Fitting model to feature number 8, Bacteroides.dorei
## 2024-04-17 18:36:08.934848 INFO::Fitting model to feature number 9, Bacteroides.eggerthii
## 2024-04-17 18:36:09.185894 INFO::Fitting model to feature number 10, Bacteroides.faecis
## 2024-04-17 18:36:09.434085 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
## 2024-04-17 18:36:09.580109 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
## 2024-04-17 18:36:09.797052 INFO::Fitting model to feature number 12, Bacteroides.fragilis
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:10.027008 INFO::Fitting model to feature number 13, Bacteroides.intestinalis
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:10.237579 INFO::Fitting model to feature number 14, Bacteroides.massiliensis
## 2024-04-17 18:36:10.439345 INFO::Fitting model to feature number 15, Bacteroides.ovatus
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:10.636809 INFO::Fitting model to feature number 16, Bacteroides.salyersiae
## 2024-04-17 18:36:10.78551 INFO::Fitting model to feature number 17, Bacteroides.stercoris
## 2024-04-17 18:36:10.946569 INFO::Fitting model to feature number 18, Bacteroides.thetaiotaomicron
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:11.110152 INFO::Fitting model to feature number 19, Bacteroides.uniformis
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:11.291426 INFO::Fitting model to feature number 20, Bacteroides.vulgatus
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:11.432675 INFO::Fitting model to feature number 21, Bacteroides.xylanisolvens
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:11.568132 INFO::Fitting model to feature number 22, Bacteroidales.bacterium.ph8
## 2024-04-17 18:36:11.725139 INFO::Fitting model to feature number 23, Barnesiella.intestinihominis
## 2024-04-17 18:36:11.960626 INFO::Fitting model to feature number 24, Coprobacter.fastidiosus
## 2024-04-17 18:36:12.116475 INFO::Fitting model to feature number 25, Odoribacter.splanchnicus
## 2024-04-17 18:36:12.526274 INFO::Fitting model to feature number 26, Parabacteroides.distasonis
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:12.65774 INFO::Fitting model to feature number 27, Parabacteroides.goldsteinii
## 2024-04-17 18:36:12.797465 INFO::Fitting model to feature number 28, Parabacteroides.merdae
## 2024-04-17 18:36:12.979242 INFO::Fitting model to feature number 29, Parabacteroides.unclassified
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:13.145385 INFO::Fitting model to feature number 30, Paraprevotella.clara
## 2024-04-17 18:36:13.370487 INFO::Fitting model to feature number 31, Paraprevotella.unclassified
## 2024-04-17 18:36:13.587579 INFO::Fitting model to feature number 32, Prevotella.copri
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:13.815829 INFO::Fitting model to feature number 33, Alistipes.finegoldii
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:14.023403 INFO::Fitting model to feature number 34, Alistipes.onderdonkii
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:14.239929 INFO::Fitting model to feature number 35, Alistipes.putredinis
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:14.43333 INFO::Fitting model to feature number 36, Alistipes.shahii
## 2024-04-17 18:36:14.639622 INFO::Fitting model to feature number 37, Alistipes.unclassified
## 2024-04-17 18:36:14.821817 INFO::Fitting model to feature number 38, Streptococcus.salivarius
## 2024-04-17 18:36:14.994641 INFO::Fitting model to feature number 39, Clostridium.bolteae
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:15.171424 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
## 2024-04-17 18:36:15.291489 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
## 2024-04-17 18:36:15.459138 INFO::Fitting model to feature number 41, Clostridium.clostridioforme
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:15.640985 INFO::Fitting model to feature number 42, Clostridium.hathewayi
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:15.848957 INFO::Fitting model to feature number 43, Clostridium.leptum
## 2024-04-17 18:36:16.077599 INFO::Fitting model to feature number 44, Clostridium.nexile
## 2024-04-17 18:36:16.285952 INFO::Fitting model to feature number 45, Clostridium.symbiosum
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:16.468347 INFO::Fitting model to feature number 46, Flavonifractor.plautii
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:16.665021 INFO::Fitting model to feature number 47, Eubacterium.eligens
## 2024-04-17 18:36:16.897855 INFO::Fitting model to feature number 48, Eubacterium.hallii
## 2024-04-17 18:36:17.133935 INFO::Fitting model to feature number 49, Eubacterium.rectale
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:17.351648 INFO::Fitting model to feature number 50, Eubacterium.siraeum
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:17.528163 INFO::Fitting model to feature number 51, Eubacterium.sp.3.1.31
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:17.741517 INFO::Fitting model to feature number 52, Eubacterium.ventriosum
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:17.937516 INFO::Fitting model to feature number 53, Ruminococcus.gnavus
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:18.175798 INFO::Fitting model to feature number 54, Ruminococcus.obeum
## 2024-04-17 18:36:18.330083 INFO::Fitting model to feature number 55, Ruminococcus.torques
## 2024-04-17 18:36:18.501316 INFO::Fitting model to feature number 56, Coprococcus.comes
## 2024-04-17 18:36:18.708811 INFO::Fitting model to feature number 57, Dorea.longicatena
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:18.933295 INFO::Fitting model to feature number 58, Lachnospiraceae.bacterium.1.1.57FAA
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:19.189619 INFO::Fitting model to feature number 59, Lachnospiraceae.bacterium.3.1.46FAA
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:19.401748 INFO::Fitting model to feature number 60, Roseburia.hominis
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:19.57704 INFO::Fitting model to feature number 61, Roseburia.intestinalis
## 2024-04-17 18:36:19.755961 INFO::Fitting model to feature number 62, Roseburia.inulinivorans
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:19.901545 INFO::Fitting model to feature number 63, Roseburia.unclassified
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:20.043362 INFO::Fitting model to feature number 64, Oscillibacter.unclassified
## 2024-04-17 18:36:20.201165 INFO::Fitting model to feature number 65, Peptostreptococcaceae.noname.unclassified
## 2024-04-17 18:36:20.342974 INFO::Fitting model to feature number 66, Faecalibacterium.prausnitzii
## 2024-04-17 18:36:20.47178 INFO::Fitting model to feature number 67, Ruminococcus.bromii
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:20.618004 INFO::Fitting model to feature number 68, Ruminococcus.callidus
## 2024-04-17 18:36:20.757058 INFO::Fitting model to feature number 69, Ruminococcus.lactaris
## 2024-04-17 18:36:20.914761 INFO::Fitting model to feature number 70, Subdoligranulum.unclassified
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:21.053513 INFO::Fitting model to feature number 71, Coprobacillus.unclassified
## 2024-04-17 18:36:21.262815 INFO::Fitting model to feature number 72, Acidaminococcus.unclassified
## 2024-04-17 18:36:21.419058 INFO::Fitting model to feature number 73, Dialister.invisus
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:21.570497 INFO::Fitting model to feature number 74, Veillonella.atypica
## 2024-04-17 18:36:21.757288 INFO::Fitting model to feature number 75, Veillonella.dispar
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:21.977973 INFO::Fitting model to feature number 76, Veillonella.parvula
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:22.210707 INFO::Fitting model to feature number 77, Veillonella.unclassified
## 2024-04-17 18:36:22.417512 INFO::Fitting model to feature number 78, Burkholderiales.bacterium.1.1.47
## 2024-04-17 18:36:22.632834 INFO::Fitting model to feature number 79, Parasutterella.excrementihominis
## 2024-04-17 18:36:22.866046 INFO::Fitting model to feature number 80, Sutterella.wadsworthensis
## 2024-04-17 18:36:23.081869 INFO::Fitting model to feature number 81, Bilophila.unclassified
## 2024-04-17 18:36:23.313862 INFO::Fitting model to feature number 82, Escherichia.coli
## 2024-04-17 18:36:23.546867 INFO::Fitting model to feature number 83, Escherichia.unclassified
## 2024-04-17 18:36:23.671942 INFO::Fitting model to feature number 84, Klebsiella.pneumoniae
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:23.8074 INFO::Fitting model to feature number 85, Haemophilus.parainfluenzae
## boundary (singular) fit: see help('isSingular')
## 2024-04-17 18:36:23.959459 INFO::Fitting model to feature number 86, Akkermansia.muciniphila
## 2024-04-17 18:36:24.1038 INFO::Fitting model to feature number 87, C2likevirus.unclassified
## 2024-04-17 18:36:24.330853 INFO::Counting total values for each feature
## 2024-04-17 18:36:24.384392 INFO::Writing filtered data to file demo_output/features/filtered_data.tsv
## 2024-04-17 18:36:24.686887 INFO::Writing filtered, normalized data to file demo_output/features/filtered_data_norm.tsv
## 2024-04-17 18:36:25.034942 INFO::Writing filtered, normalized, transformed data to file demo_output/features/filtered_data_norm_transformed.tsv
## 2024-04-17 18:36:25.575883 INFO::Writing residuals to file demo_output/fits/residuals.rds
## 2024-04-17 18:36:25.643422 INFO::Writing fitted values to file demo_output/fits/fitted.rds
## 2024-04-17 18:36:25.681218 INFO::Writing extracted random effects to file demo_output/fits/ranef.rds
## 2024-04-17 18:36:25.68967 INFO::Writing all results to file (ordered by increasing q-values): demo_output/all_results.tsv
## 2024-04-17 18:36:25.706509 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
## 2024-04-17 18:36:25.715165 INFO::Writing heatmap of significant results to file: demo_output/heatmap.pdf
## 2024-04-17 18:36:25.950245 INFO::Writing association plots (one for each significant association) to output folder: demo_output
## 2024-04-17 18:36:25.959705 INFO::Plotting associations from most to least significant, grouped by metadata
## 2024-04-17 18:36:25.961842 INFO::Plotting data for metadata number 1, dysbiosisCD
## 2024-04-17 18:36:25.964767 INFO::Creating boxplot for categorical data, dysbiosisCD vs Faecalibacterium.prausnitzii
## 2024-04-17 18:36:26.382799 INFO::Creating boxplot for categorical data, dysbiosisCD vs Subdoligranulum.unclassified
## 2024-04-17 18:36:26.691267 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.uniformis
## 2024-04-17 18:36:26.981078 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.rectale
## 2024-04-17 18:36:27.412406 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.vulgatus
## 2024-04-17 18:36:27.739912 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.ovatus
## 2024-04-17 18:36:28.130283 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.obeum
## 2024-04-17 18:36:28.484882 INFO::Creating boxplot for categorical data, dysbiosisCD vs Oscillibacter.unclassified
## 2024-04-17 18:36:28.775575 INFO::Creating boxplot for categorical data, dysbiosisCD vs Roseburia.inulinivorans
## 2024-04-17 18:36:29.135694 INFO::Creating boxplot for categorical data, dysbiosisCD vs Roseburia.hominis
## 2024-04-17 18:36:29.424837 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.thetaiotaomicron
## 2024-04-17 18:36:29.686785 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.putredinis
## 2024-04-17 18:36:29.956336 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.distasonis
## 2024-04-17 18:36:30.211995 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.dorei
## 2024-04-17 18:36:30.474607 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.shahii
## 2024-04-17 18:36:30.761094 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.xylanisolvens
## 2024-04-17 18:36:31.087274 INFO::Creating boxplot for categorical data, dysbiosisCD vs Clostridium.leptum
## 2024-04-17 18:36:31.470665 INFO::Creating boxplot for categorical data, dysbiosisCD vs Dorea.longicatena
## 2024-04-17 18:36:31.868529 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.caccae
## 2024-04-17 18:36:32.279061 INFO::Creating boxplot for categorical data, dysbiosisCD vs Lachnospiraceae.bacterium.3.1.46FAA
## 2024-04-17 18:36:32.548762 INFO::Creating boxplot for categorical data, dysbiosisCD vs Escherichia.coli
## 2024-04-17 18:36:32.833124 INFO::Creating boxplot for categorical data, dysbiosisCD vs Klebsiella.pneumoniae
## 2024-04-17 18:36:33.704862 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bilophila.unclassified
## 2024-04-17 18:36:33.979464 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.finegoldii
## 2024-04-17 18:36:34.317441 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.eligens
## 2024-04-17 18:36:34.599032 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.stercoris
## 2024-04-17 18:36:34.967003 INFO::Creating boxplot for categorical data, dysbiosisCD vs Coprococcus.comes
## 2024-04-17 18:36:35.295997 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.fragilis
## 2024-04-17 18:36:35.691009 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.finegoldii
## 2024-04-17 18:36:35.93962 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.hallii
## 2024-04-17 18:36:36.215272 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.faecis
## 2024-04-17 18:36:36.488645 INFO::Creating boxplot for categorical data, dysbiosisCD vs Paraprevotella.clara
## 2024-04-17 18:36:36.753534 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.siraeum
## 2024-04-17 18:36:37.142824 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.merdae
## 2024-04-17 18:36:37.442311 INFO::Creating boxplot for categorical data, dysbiosisCD vs Paraprevotella.unclassified
## 2024-04-17 18:36:37.739106 INFO::Creating boxplot for categorical data, dysbiosisCD vs Collinsella.aerofaciens
## 2024-04-17 18:36:38.062963 INFO::Creating boxplot for categorical data, dysbiosisCD vs Odoribacter.splanchnicus
## 2024-04-17 18:36:38.323951 INFO::Creating boxplot for categorical data, dysbiosisCD vs Clostridium.clostridioforme
## 2024-04-17 18:36:38.563489 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.eggerthii
## 2024-04-17 18:36:38.82928 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.onderdonkii
## 2024-04-17 18:36:39.062984 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.ventriosum
## 2024-04-17 18:36:39.353762 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.lactaris
## 2024-04-17 18:36:39.594775 INFO::Creating boxplot for categorical data, dysbiosisCD vs Burkholderiales.bacterium.1.1.47
## 2024-04-17 18:36:39.854015 INFO::Creating boxplot for categorical data, dysbiosisCD vs Dialister.invisus
## 2024-04-17 18:36:40.1325 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.bromii
## 2024-04-17 18:36:40.557875 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parasutterella.excrementihominis
## 2024-04-17 18:36:40.956642 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.unclassified
## 2024-04-17 18:36:41.262512 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.torques
## 2024-04-17 18:36:41.563712 INFO::Creating boxplot for categorical data, dysbiosisCD vs Coprobacillus.unclassified
## 2024-04-17 18:36:41.916281 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.cellulosilyticus
## 2024-04-17 18:36:42.152156 INFO::Creating boxplot for categorical data, dysbiosisCD vs Roseburia.intestinalis
## 2024-04-17 18:36:42.407289 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.unclassified
## 2024-04-17 18:36:42.681412 INFO::Creating boxplot for categorical data, dysbiosisCD vs Acidaminococcus.unclassified
## 2024-04-17 18:36:42.985142 INFO::Creating boxplot for categorical data, dysbiosisCD vs Barnesiella.intestinihominis
## 2024-04-17 18:36:43.24371 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.massiliensis
## 2024-04-17 18:36:43.531199 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.sp.3.1.31
## 2024-04-17 18:36:43.789827 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.salyersiae
## 2024-04-17 18:36:44.051313 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroidales.bacterium.ph8
## 2024-04-17 18:36:44.30024 INFO::Creating boxplot for categorical data, dysbiosisCD vs Clostridium.citroniae
## 2024-04-17 18:36:44.629568 INFO::Creating boxplot for categorical data, dysbiosisCD vs Flavonifractor.plautii
## 2024-04-17 18:36:45.018073 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.goldsteinii
## 2024-04-17 18:36:45.356735 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bifidobacterium.longum
## 2024-04-17 18:36:45.620126 INFO::Creating boxplot for categorical data, dysbiosisCD vs Lachnospiraceae.bacterium.1.1.57FAA
## 2024-04-17 18:36:49.943933 INFO::Plotting data for metadata number 2, dysbiosisUC
## 2024-04-17 18:36:49.947084 INFO::Creating boxplot for categorical data, dysbiosisUC vs Subdoligranulum.unclassified
## 2024-04-17 18:36:50.652755 INFO::Creating boxplot for categorical data, dysbiosisUC vs Faecalibacterium.prausnitzii
## 2024-04-17 18:36:51.016156 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.caccae
## 2024-04-17 18:36:51.435889 INFO::Creating boxplot for categorical data, dysbiosisUC vs Oscillibacter.unclassified
## 2024-04-17 18:36:51.774732 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.uniformis
## 2024-04-17 18:36:52.179283 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.siraeum
## 2024-04-17 18:36:52.526265 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.ovatus
## 2024-04-17 18:36:52.878847 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.fragilis
## 2024-04-17 18:36:53.160321 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.shahii
## 2024-04-17 18:36:53.495703 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.rectale
## 2024-04-17 18:36:53.77572 INFO::Creating boxplot for categorical data, dysbiosisUC vs Roseburia.hominis
## 2024-04-17 18:36:54.028464 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.putredinis
## 2024-04-17 18:36:54.29869 INFO::Creating boxplot for categorical data, dysbiosisUC vs Lachnospiraceae.bacterium.3.1.46FAA
## 2024-04-17 18:36:54.703059 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.hallii
## 2024-04-17 18:36:55.036794 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.xylanisolvens
## 2024-04-17 18:36:55.314744 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.stercoris
## 2024-04-17 18:36:55.579543 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.finegoldii
## 2024-04-17 18:36:55.845391 INFO::Creating boxplot for categorical data, dysbiosisUC vs Barnesiella.intestinihominis
## 2024-04-17 18:36:56.165363 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.leptum
## 2024-04-17 18:36:56.431153 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.gnavus
## 2024-04-17 18:36:56.778059 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.onderdonkii
## 2024-04-17 18:36:57.028352 INFO::Creating boxplot for categorical data, dysbiosisUC vs Flavonifractor.plautii
## 2024-04-17 18:36:57.345654 INFO::Creating boxplot for categorical data, dysbiosisUC vs Parabacteroides.merdae
## 2024-04-17 18:36:57.725643 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.cellulosilyticus
## 2024-04-17 18:36:58.058515 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bifidobacterium.longum
## 2024-04-17 18:36:58.319333 INFO::Creating boxplot for categorical data, dysbiosisUC vs Klebsiella.pneumoniae
## 2024-04-17 18:36:58.619725 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.citroniae
## 2024-04-17 18:36:59.022327 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.ventriosum
## 2024-04-17 18:36:59.392104 INFO::Creating boxplot for categorical data, dysbiosisUC vs Parabacteroides.distasonis
## 2024-04-17 18:36:59.756018 INFO::Creating boxplot for categorical data, dysbiosisUC vs Parabacteroides.goldsteinii
## 2024-04-17 18:37:00.025773 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.torques
## 2024-04-17 18:37:00.297712 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroidales.bacterium.ph8
## 2024-04-17 18:37:00.724377 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.obeum
## 2024-04-17 18:37:01.033753 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bifidobacterium.bifidum
## 2024-04-17 18:37:01.426184 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bifidobacterium.adolescentis
## 2024-04-17 18:37:01.738709 INFO::Creating boxplot for categorical data, dysbiosisUC vs Collinsella.aerofaciens
## 2024-04-17 18:37:02.048637 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.hathewayi
## 2024-04-17 18:37:02.418401 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bilophila.unclassified
## 2024-04-17 18:37:02.735826 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.eligens
## 2024-04-17 18:37:03.08 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.vulgatus
## 2024-04-17 18:37:03.41934 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.bolteae
## 2024-04-17 18:37:03.796328 INFO::Creating boxplot for categorical data, dysbiosisUC vs Dialister.invisus
## 2024-04-17 18:37:04.176053 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.lactaris
## 2024-04-17 18:37:04.590447 INFO::Creating boxplot for categorical data, dysbiosisUC vs Burkholderiales.bacterium.1.1.47
## 2024-04-17 18:37:08.980661 INFO::Plotting data for metadata number 3, dysbiosisnonIBD
## 2024-04-17 18:37:08.985376 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Faecalibacterium.prausnitzii
## 2024-04-17 18:37:09.248623 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.rectale
## 2024-04-17 18:37:09.522214 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.sp.3.1.31
## 2024-04-17 18:37:09.824297 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.hominis
## 2024-04-17 18:37:10.129043 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Subdoligranulum.unclassified
## 2024-04-17 18:37:10.451745 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Lachnospiraceae.bacterium.3.1.46FAA
## 2024-04-17 18:37:10.73714 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Ruminococcus.torques
## 2024-04-17 18:37:11.046702 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Clostridium.leptum
## 2024-04-17 18:37:11.420001 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Odoribacter.splanchnicus
## 2024-04-17 18:37:11.850892 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Escherichia.coli
## 2024-04-17 18:37:12.243539 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Ruminococcus.obeum
## 2024-04-17 18:37:12.666565 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bilophila.unclassified
## 2024-04-17 18:37:13.066168 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.uniformis
## 2024-04-17 18:37:13.452864 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.eligens
## 2024-04-17 18:37:13.78338 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Klebsiella.pneumoniae
## 2024-04-17 18:37:14.079451 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Ruminococcus.bromii
## 2024-04-17 18:37:14.403861 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.vulgatus
## 2024-04-17 18:37:14.820958 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs C2likevirus.unclassified
## 2024-04-17 18:37:15.136788 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bifidobacterium.adolescentis
## 2024-04-17 18:37:15.430543 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Alistipes.finegoldii
## 2024-04-17 18:37:15.720147 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.inulinivorans
## 2024-04-17 18:37:16.010481 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Alistipes.onderdonkii
## 2024-04-17 18:37:16.298524 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.unclassified
## 2024-04-17 18:37:16.580172 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.hallii
## 2024-04-17 18:37:16.880246 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.intestinalis
## 2024-04-17 18:37:17.198005 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Prevotella.copri
## 2024-04-17 18:37:17.578484 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.dorei
## 2024-04-17 18:37:17.940799 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.fragilis
## 2024-04-17 18:37:18.22801 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Alistipes.shahii
## 2024-04-17 18:37:22.817363 INFO::Plotting data for metadata number 4, antibiotics
## 2024-04-17 18:37:22.820647 INFO::Creating boxplot for categorical data, antibiotics vs Roseburia.inulinivorans
## 2024-04-17 18:37:23.073937 INFO::Creating boxplot for categorical data, antibiotics vs Roseburia.hominis
## 2024-04-17 18:37:23.339824 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.rectale
## 2024-04-17 18:37:23.619343 INFO::Creating boxplot for categorical data, antibiotics vs Dialister.invisus
## 2024-04-17 18:37:23.955624 INFO::Creating boxplot for categorical data, antibiotics vs Roseburia.intestinalis
## 2024-04-17 18:37:24.216772 INFO::Creating boxplot for categorical data, antibiotics vs Dorea.longicatena
## 2024-04-17 18:37:24.489633 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.callidus
## 2024-04-17 18:37:24.784065 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.bromii
## 2024-04-17 18:37:25.1018 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.obeum
## 2024-04-17 18:37:25.493486 INFO::Creating boxplot for categorical data, antibiotics vs Klebsiella.pneumoniae
## 2024-04-17 18:37:25.888279 INFO::Creating boxplot for categorical data, antibiotics vs Bifidobacterium.adolescentis
## 2024-04-17 18:37:26.181297 INFO::Creating boxplot for categorical data, antibiotics vs Faecalibacterium.prausnitzii
## 2024-04-17 18:37:26.565978 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.hallii
## 2024-04-17 18:37:26.868271 INFO::Creating boxplot for categorical data, antibiotics vs Bilophila.unclassified
## 2024-04-17 18:37:27.198239 INFO::Creating boxplot for categorical data, antibiotics vs Clostridium.leptum
## 2024-04-17 18:37:27.535214 INFO::Creating boxplot for categorical data, antibiotics vs Lachnospiraceae.bacterium.3.1.46FAA
## 2024-04-17 18:37:27.936094 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.finegoldii
## 2024-04-17 18:37:28.196109 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.sp.3.1.31
## 2024-04-17 18:37:28.448496 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.fragilis
## 2024-04-17 18:37:28.702405 INFO::Creating boxplot for categorical data, antibiotics vs Alistipes.onderdonkii
## 2024-04-17 18:37:29.013689 INFO::Creating boxplot for categorical data, antibiotics vs Sutterella.wadsworthensis
## 2024-04-17 18:37:29.31585 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.eligens
## 2024-04-17 18:37:29.560663 INFO::Creating boxplot for categorical data, antibiotics vs Collinsella.aerofaciens
## 2024-04-17 18:37:29.846881 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.thetaiotaomicron
## 2024-04-17 18:37:30.100325 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.eggerthii
## 2024-04-17 18:37:30.470441 INFO::Creating boxplot for categorical data, antibiotics vs Haemophilus.parainfluenzae
## 2024-04-17 18:37:30.865097 INFO::Creating boxplot for categorical data, antibiotics vs Bifidobacterium.pseudocatenulatum
## 2024-04-17 18:37:31.218663 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.torques
## 2024-04-17 18:37:31.550664 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.ventriosum
## 2024-04-17 18:37:31.895582 INFO::Creating boxplot for categorical data, antibiotics vs Parasutterella.excrementihominis
## 2024-04-17 18:37:32.199472 INFO::Creating boxplot for categorical data, antibiotics vs Peptostreptococcaceae.noname.unclassified
## 2024-04-17 18:37:32.540454 INFO::Creating boxplot for categorical data, antibiotics vs Veillonella.dispar
## 2024-04-17 18:37:32.84009 INFO::Creating boxplot for categorical data, antibiotics vs Veillonella.atypica
## 2024-04-17 18:37:33.110379 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroidales.bacterium.ph8
## 2024-04-17 18:37:33.415809 INFO::Creating boxplot for categorical data, antibiotics vs Clostridium.nexile
## 2024-04-17 18:37:33.760378 INFO::Creating boxplot for categorical data, antibiotics vs Burkholderiales.bacterium.1.1.47
## 2024-04-17 18:37:34.111531 INFO::Creating boxplot for categorical data, antibiotics vs Lachnospiraceae.bacterium.1.1.57FAA
## 2024-04-17 18:37:34.502027 INFO::Creating boxplot for categorical data, antibiotics vs Akkermansia.muciniphila
## 2024-04-17 18:37:34.876326 INFO::Creating boxplot for categorical data, antibiotics vs Clostridium.citroniae
## 2024-04-17 18:37:35.227463 INFO::Creating boxplot for categorical data, antibiotics vs Odoribacter.splanchnicus
## 2024-04-17 18:37:39.758237 INFO::Plotting data for metadata number 5, age
## 2024-04-17 18:37:39.761464 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()`).
## 2024-04-17 18:37:40.172826 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()`).
## 2024-04-17 18:37:40.707971 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()`).
## 2024-04-17 18:37:41.481583 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()`).
## 2024-04-17 18:37:41.954094 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()`).
## 2024-04-17 18:37:42.356213 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()`).
## 2024-04-17 18:37:42.722532 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()`).
## 2024-04-17 18:37:43.037564 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()`).
## 2024-04-17 18:37:43.362529 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()`).
## 2024-04-17 18:37:43.703417 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()`).
## 2024-04-17 18:37:44.115809 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()`).
## 2024-04-17 18:37:44.544038 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()`).
## 2024-04-17 18:37:44.942318 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()`).
## 2024-04-17 18:37:45.334434 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()`).
## 2024-04-17 18:37:45.606074 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()`).
## 2024-04-17 18:37:45.93602 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()`).
## 2024-04-17 18:37:46.287328 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()`).
## 2024-04-17 18:37:46.580779 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()`).
## 2024-04-17 18:37:46.947053 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()`).
## 2024-04-17 18:37:47.29914 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()`).
## 2024-04-17 18:37:47.642296 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()`).
## 2024-04-17 18:37:47.95353 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()`).
## 2024-04-17 18:37:52.452816 INFO::Plotting data for metadata number 6, diagnosis
## 2024-04-17 18:37:52.455843 INFO::Creating boxplot for categorical data, diagnosis vs Bifidobacterium.adolescentis
## 2024-04-17 18:37:52.696 INFO::Creating boxplot for categorical data, diagnosis vs Akkermansia.muciniphila
## 2024-04-17 18:37:52.950347 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.bolteae
## 2024-04-17 18:37:53.200575 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.putredinis
## 2024-04-17 18:37:53.463719 INFO::Creating boxplot for categorical data, diagnosis vs Coprobacillus.unclassified
## 2024-04-17 18:37:53.72769 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.clostridioforme
## 2024-04-17 18:37:53.977912 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.shahii
## 2024-04-17 18:37:54.248015 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.bromii
## 2024-04-17 18:37:54.563926 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.leptum
## 2024-04-17 18:37:54.828831 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.symbiosum
## 2024-04-17 18:37:55.093232 INFO::Creating boxplot for categorical data, diagnosis vs Roseburia.inulinivorans
## 2024-04-17 18:37:55.394502 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.bromii
## 2024-04-17 18:37:55.702726 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.shahii
## 2024-04-17 18:37:55.956131 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.finegoldii
## 2024-04-17 18:37:56.20707 INFO::Creating boxplot for categorical data, diagnosis vs Parabacteroides.goldsteinii
## 2024-04-17 18:37:56.526882 INFO::Creating boxplot for categorical data, diagnosis vs Bilophila.unclassified
## 2024-04-17 18:37:56.851927 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.leptum
## 2024-04-17 18:37:57.236236 INFO::Creating boxplot for categorical data, diagnosis vs Roseburia.hominis
## 2024-04-17 18:37:57.501309 INFO::Creating boxplot for categorical data, diagnosis vs Coprobacillus.unclassified
## 2024-04-17 18:37:57.760258 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.onderdonkii
## 2024-04-17 18:37:58.011435 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.ventriosum
## 2024-04-17 18:37:58.28239 INFO::Creating boxplot for categorical data, diagnosis vs Roseburia.hominis
## 2024-04-17 18:37:58.531948 INFO::Creating boxplot for categorical data, diagnosis vs Sutterella.wadsworthensis
## 2024-04-17 18:37:58.803944 INFO::Creating boxplot for categorical data, diagnosis vs Akkermansia.muciniphila
## 2024-04-17 18:37:59.061194 INFO::Creating boxplot for categorical data, diagnosis vs Sutterella.wadsworthensis
## 2024-04-17 18:37:59.32066 INFO::Creating boxplot for categorical data, diagnosis vs Subdoligranulum.unclassified
## 2024-04-17 18:37:59.57064 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.symbiosum
## 2024-04-17 18:37:59.831405 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.onderdonkii
## 2024-04-17 18:38:00.080604 INFO::Creating boxplot for categorical data, diagnosis vs Parabacteroides.unclassified
## 2024-04-17 18:38:00.334302 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.lactaris
## 2024-04-17 18:38:00.574193 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.callidus
## 2024-04-17 18:38:00.845208 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.gnavus
## 2024-04-17 18:38:01.152881 INFO::Creating boxplot for categorical data, diagnosis vs Bacteroides.fragilis
## 2024-04-17 18:38:01.505383 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.rectale
## 2024-04-17 18:38:01.762904 INFO::Creating boxplot for categorical data, diagnosis vs Odoribacter.splanchnicus
## 2024-04-17 18:38:02.014531 INFO::Creating boxplot for categorical data, diagnosis vs Parabacteroides.distasonis
## 2024-04-17 18:38:02.28359 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.rectale
## 2024-04-17 18:38:02.548626 INFO::Creating boxplot for categorical data, diagnosis vs Bacteroides.finegoldii
## 2024-04-17 18:38:02.872213 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.callidus
## 2024-04-17 18:38:03.118597 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.finegoldii
## 2024-04-17 18:38:03.537788 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.putredinis
## 2024-04-17 18:38:03.790657 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.bolteae
## 2024-04-17 18:38:04.15819 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 version 4.4.0 beta (2024-04-15 r86425 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_1.17.0
## 
## loaded via a namespace (and not attached):
##  [1] gtable_0.3.4        biglm_0.9-2.1       xfun_0.43          
##  [4] bslib_0.7.0         ggplot2_3.5.0       lattice_0.22-6     
##  [7] numDeriv_2016.8-1.1 vctrs_0.6.5         tools_4.4.0        
## [10] generics_0.1.3      parallel_4.4.0      getopt_1.20.4      
## [13] tibble_3.2.1        fansi_1.0.6         DEoptimR_1.1-3     
## [16] cluster_2.1.6       pkgconfig_2.0.3     logging_0.10-108   
## [19] pheatmap_1.0.12     Matrix_1.7-0        data.table_1.15.4  
## [22] RColorBrewer_1.1-3  lifecycle_1.0.4     farver_2.1.1       
## [25] compiler_4.4.0      munsell_0.5.1       lmerTest_3.1-3     
## [28] permute_0.9-7       htmltools_0.5.8.1   sass_0.4.9         
## [31] hash_2.2.6.3        yaml_2.3.8          pillar_1.9.0       
## [34] nloptr_2.0.3        crayon_1.5.2        jquerylib_0.1.4    
## [37] MASS_7.3-60.2       cachem_1.0.8        vegan_2.6-4        
## [40] boot_1.3-30         nlme_3.1-164        robustbase_0.99-2  
## [43] tidyselect_1.2.1    digest_0.6.35       mvtnorm_1.2-4      
## [46] dplyr_1.1.4         labeling_0.4.3      splines_4.4.0      
## [49] pcaPP_2.0-4         fastmap_1.1.1       grid_4.4.0         
## [52] colorspace_2.1-0    cli_3.6.2           magrittr_2.0.3     
## [55] utf8_1.2.4          withr_3.0.0         scales_1.3.0       
## [58] rmarkdown_2.26      lme4_1.1-35.3       pbapply_1.7-2      
## [61] evaluate_0.23       knitr_1.46          mgcv_1.9-1         
## [64] rlang_1.1.3         Rcpp_1.0.12         glue_1.7.0         
## [67] optparse_1.7.5      DBI_1.2.2           minqa_1.2.6        
## [70] jsonlite_1.8.8      R6_2.5.1

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.