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"
## 2023-06-08 17:09:25.220115 INFO::Writing function arguments to log file
## 2023-06-08 17:09:25.242982 INFO::Verifying options selected are valid
## 2023-06-08 17:09:25.277342 INFO::Determining format of input files
## 2023-06-08 17:09:25.278478 INFO::Input format is data samples as rows and metadata samples as rows
## 2023-06-08 17:09:25.283217 INFO::Formula for random effects: expr ~ (1 | site) + (1 | subject)
## 2023-06-08 17:09:25.284223 INFO::Formula for fixed effects: expr ~  diagnosis + dysbiosisnonIBD + dysbiosisUC + dysbiosisCD + antibiotics + age
## 2023-06-08 17:09:25.286348 INFO::Filter data based on min abundance and min prevalence
## 2023-06-08 17:09:25.286988 INFO::Total samples in data: 1595
## 2023-06-08 17:09:25.287585 INFO::Min samples required with min abundance for a feature not to be filtered: 159.500000
## 2023-06-08 17:09:25.292371 INFO::Total filtered features: 0
## 2023-06-08 17:09:25.29316 INFO::Filtered feature names from abundance and prevalence filtering:
## 2023-06-08 17:09:25.301321 INFO::Total filtered features with variance filtering: 0
## 2023-06-08 17:09:25.302068 INFO::Filtered feature names from variance filtering:
## 2023-06-08 17:09:25.302686 INFO::Running selected normalization method: TSS
## 2023-06-08 17:09:26.582129 INFO::Bypass z-score application to metadata
## 2023-06-08 17:09:26.583501 INFO::Running selected transform method: LOG
## 2023-06-08 17:09:26.601129 INFO::Running selected analysis method: LM
## 2023-06-08 17:09:27.163928 INFO::Fitting model to feature number 1, Bifidobacterium.adolescentis
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:27.572282 INFO::Fitting model to feature number 2, Bifidobacterium.bifidum
## 2023-06-08 17:09:27.747658 INFO::Fitting model to feature number 3, Bifidobacterium.longum
## 2023-06-08 17:09:27.911273 INFO::Fitting model to feature number 4, Bifidobacterium.pseudocatenulatum
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:28.054029 INFO::Fitting model to feature number 5, Collinsella.aerofaciens
## 2023-06-08 17:09:28.21343 INFO::Fitting model to feature number 6, Bacteroides.caccae
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:28.372435 INFO::Fitting model to feature number 7, Bacteroides.cellulosilyticus
## 2023-06-08 17:09:28.531709 INFO::Fitting model to feature number 8, Bacteroides.dorei
## 2023-06-08 17:09:28.733425 INFO::Fitting model to feature number 9, Bacteroides.eggerthii
## 2023-06-08 17:09:28.930035 INFO::Fitting model to feature number 10, Bacteroides.faecis
## 2023-06-08 17:09:29.10057 INFO::Fitting model to feature number 11, Bacteroides.finegoldii
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:29.247422 INFO::Fitting model to feature number 12, Bacteroides.fragilis
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:29.394974 INFO::Fitting model to feature number 13, Bacteroides.intestinalis
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:29.549295 INFO::Fitting model to feature number 14, Bacteroides.massiliensis
## 2023-06-08 17:09:29.693033 INFO::Fitting model to feature number 15, Bacteroides.ovatus
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:29.831499 INFO::Fitting model to feature number 16, Bacteroides.salyersiae
## 2023-06-08 17:09:29.978352 INFO::Fitting model to feature number 17, Bacteroides.stercoris
## 2023-06-08 17:09:30.146859 INFO::Fitting model to feature number 18, Bacteroides.thetaiotaomicron
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:30.321844 INFO::Fitting model to feature number 19, Bacteroides.uniformis
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:30.480343 INFO::Fitting model to feature number 20, Bacteroides.vulgatus
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:30.66681 INFO::Fitting model to feature number 21, Bacteroides.xylanisolvens
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:30.882246 INFO::Fitting model to feature number 22, Bacteroidales.bacterium.ph8
## 2023-06-08 17:09:31.082798 INFO::Fitting model to feature number 23, Barnesiella.intestinihominis
## 2023-06-08 17:09:31.330229 INFO::Fitting model to feature number 24, Coprobacter.fastidiosus
## 2023-06-08 17:09:31.482838 INFO::Fitting model to feature number 25, Odoribacter.splanchnicus
## 2023-06-08 17:09:31.628506 INFO::Fitting model to feature number 26, Parabacteroides.distasonis
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:31.783385 INFO::Fitting model to feature number 27, Parabacteroides.goldsteinii
## 2023-06-08 17:09:31.93171 INFO::Fitting model to feature number 28, Parabacteroides.merdae
## 2023-06-08 17:09:32.082933 INFO::Fitting model to feature number 29, Parabacteroides.unclassified
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:32.230452 INFO::Fitting model to feature number 30, Paraprevotella.clara
## 2023-06-08 17:09:32.393156 INFO::Fitting model to feature number 31, Paraprevotella.unclassified
## 2023-06-08 17:09:32.546321 INFO::Fitting model to feature number 32, Prevotella.copri
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:32.699408 INFO::Fitting model to feature number 33, Alistipes.finegoldii
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:32.850449 INFO::Fitting model to feature number 34, Alistipes.onderdonkii
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:33.008554 INFO::Fitting model to feature number 35, Alistipes.putredinis
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:33.158445 INFO::Fitting model to feature number 36, Alistipes.shahii
## 2023-06-08 17:09:33.336263 INFO::Fitting model to feature number 37, Alistipes.unclassified
## 2023-06-08 17:09:33.492275 INFO::Fitting model to feature number 38, Streptococcus.salivarius
## 2023-06-08 17:09:33.646565 INFO::Fitting model to feature number 39, Clostridium.bolteae
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:33.810392 INFO::Fitting model to feature number 40, Clostridium.citroniae
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:33.957536 INFO::Fitting model to feature number 41, Clostridium.clostridioforme
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:34.104944 INFO::Fitting model to feature number 42, Clostridium.hathewayi
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:34.256175 INFO::Fitting model to feature number 43, Clostridium.leptum
## 2023-06-08 17:09:34.413762 INFO::Fitting model to feature number 44, Clostridium.nexile
## 2023-06-08 17:09:34.568976 INFO::Fitting model to feature number 45, Clostridium.symbiosum
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:34.738185 INFO::Fitting model to feature number 46, Flavonifractor.plautii
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:34.891335 INFO::Fitting model to feature number 47, Eubacterium.eligens
## 2023-06-08 17:09:35.033503 INFO::Fitting model to feature number 48, Eubacterium.hallii
## 2023-06-08 17:09:35.187999 INFO::Fitting model to feature number 49, Eubacterium.rectale
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:35.348254 INFO::Fitting model to feature number 50, Eubacterium.siraeum
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:35.50701 INFO::Fitting model to feature number 51, Eubacterium.sp.3.1.31
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:35.65294 INFO::Fitting model to feature number 52, Eubacterium.ventriosum
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:35.792278 INFO::Fitting model to feature number 53, Ruminococcus.gnavus
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:35.94339 INFO::Fitting model to feature number 54, Ruminococcus.obeum
## 2023-06-08 17:09:36.086302 INFO::Fitting model to feature number 55, Ruminococcus.torques
## 2023-06-08 17:09:36.238291 INFO::Fitting model to feature number 56, Coprococcus.comes
## 2023-06-08 17:09:36.38471 INFO::Fitting model to feature number 57, Dorea.longicatena
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:36.542211 INFO::Fitting model to feature number 58, Lachnospiraceae.bacterium.1.1.57FAA
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:36.715451 INFO::Fitting model to feature number 59, Lachnospiraceae.bacterium.3.1.46FAA
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:36.867582 INFO::Fitting model to feature number 60, Roseburia.hominis
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:37.020498 INFO::Fitting model to feature number 61, Roseburia.intestinalis
## 2023-06-08 17:09:37.157959 INFO::Fitting model to feature number 62, Roseburia.inulinivorans
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:37.306702 INFO::Fitting model to feature number 63, Roseburia.unclassified
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:37.451819 INFO::Fitting model to feature number 64, Oscillibacter.unclassified
## 2023-06-08 17:09:37.618941 INFO::Fitting model to feature number 65, Peptostreptococcaceae.noname.unclassified
## 2023-06-08 17:09:37.7637 INFO::Fitting model to feature number 66, Faecalibacterium.prausnitzii
## 2023-06-08 17:09:37.915005 INFO::Fitting model to feature number 67, Ruminococcus.bromii
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:38.051786 INFO::Fitting model to feature number 68, Ruminococcus.callidus
## 2023-06-08 17:09:38.197835 INFO::Fitting model to feature number 69, Ruminococcus.lactaris
## 2023-06-08 17:09:38.599224 INFO::Fitting model to feature number 70, Subdoligranulum.unclassified
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:38.742882 INFO::Fitting model to feature number 71, Coprobacillus.unclassified
## 2023-06-08 17:09:38.878906 INFO::Fitting model to feature number 72, Acidaminococcus.unclassified
## 2023-06-08 17:09:39.035563 INFO::Fitting model to feature number 73, Dialister.invisus
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:39.189482 INFO::Fitting model to feature number 74, Veillonella.atypica
## 2023-06-08 17:09:39.356256 INFO::Fitting model to feature number 75, Veillonella.dispar
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:39.499655 INFO::Fitting model to feature number 76, Veillonella.parvula
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:39.64159 INFO::Fitting model to feature number 77, Veillonella.unclassified
## 2023-06-08 17:09:39.77847 INFO::Fitting model to feature number 78, Burkholderiales.bacterium.1.1.47
## 2023-06-08 17:09:39.928995 INFO::Fitting model to feature number 79, Parasutterella.excrementihominis
## 2023-06-08 17:09:40.067917 INFO::Fitting model to feature number 80, Sutterella.wadsworthensis
## 2023-06-08 17:09:40.211323 INFO::Fitting model to feature number 81, Bilophila.unclassified
## 2023-06-08 17:09:40.359599 INFO::Fitting model to feature number 82, Escherichia.coli
## 2023-06-08 17:09:40.514378 INFO::Fitting model to feature number 83, Escherichia.unclassified
## 2023-06-08 17:09:40.668615 INFO::Fitting model to feature number 84, Klebsiella.pneumoniae
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:40.814076 INFO::Fitting model to feature number 85, Haemophilus.parainfluenzae
## boundary (singular) fit: see help('isSingular')
## 2023-06-08 17:09:40.958768 INFO::Fitting model to feature number 86, Akkermansia.muciniphila
## 2023-06-08 17:09:41.105356 INFO::Fitting model to feature number 87, C2likevirus.unclassified
## 2023-06-08 17:09:41.297481 INFO::Counting total values for each feature
## 2023-06-08 17:09:41.328439 INFO::Writing filtered data to file demo_output/features/filtered_data.tsv
## 2023-06-08 17:09:41.468234 INFO::Writing filtered, normalized data to file demo_output/features/filtered_data_norm.tsv
## 2023-06-08 17:09:41.609308 INFO::Writing filtered, normalized, transformed data to file demo_output/features/filtered_data_norm_transformed.tsv
## 2023-06-08 17:09:41.831069 INFO::Writing residuals to file demo_output/fits/residuals.rds
## 2023-06-08 17:09:41.892409 INFO::Writing fitted values to file demo_output/fits/fitted.rds
## 2023-06-08 17:09:41.926727 INFO::Writing extracted random effects to file demo_output/fits/ranef.rds
## 2023-06-08 17:09:41.932026 INFO::Writing all results to file (ordered by increasing q-values): demo_output/all_results.tsv
## 2023-06-08 17:09:41.937925 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
## 2023-06-08 17:09:41.94063 INFO::Writing heatmap of significant results to file: demo_output/heatmap.pdf
## 2023-06-08 17:09:42.155949 INFO::Writing association plots (one for each significant association) to output folder: demo_output
## 2023-06-08 17:09:42.16159 INFO::Plotting associations from most to least significant, grouped by metadata
## 2023-06-08 17:09:42.162479 INFO::Plotting data for metadata number 1, dysbiosisCD
## 2023-06-08 17:09:42.163903 INFO::Creating boxplot for categorical data, dysbiosisCD vs Faecalibacterium.prausnitzii
## 2023-06-08 17:09:42.518252 INFO::Creating boxplot for categorical data, dysbiosisCD vs Subdoligranulum.unclassified
## 2023-06-08 17:09:42.766398 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.uniformis
## 2023-06-08 17:09:43.012599 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.rectale
## 2023-06-08 17:09:43.262853 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.vulgatus
## 2023-06-08 17:09:43.496278 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.ovatus
## 2023-06-08 17:09:43.736661 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.obeum
## 2023-06-08 17:09:43.968716 INFO::Creating boxplot for categorical data, dysbiosisCD vs Oscillibacter.unclassified
## 2023-06-08 17:09:44.219476 INFO::Creating boxplot for categorical data, dysbiosisCD vs Roseburia.inulinivorans
## 2023-06-08 17:09:44.46325 INFO::Creating boxplot for categorical data, dysbiosisCD vs Roseburia.hominis
## 2023-06-08 17:09:44.713243 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.thetaiotaomicron
## 2023-06-08 17:09:44.949218 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.putredinis
## 2023-06-08 17:09:45.189331 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.distasonis
## 2023-06-08 17:09:45.429672 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.dorei
## 2023-06-08 17:09:45.661002 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.shahii
## 2023-06-08 17:09:45.901708 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.xylanisolvens
## 2023-06-08 17:09:46.130773 INFO::Creating boxplot for categorical data, dysbiosisCD vs Clostridium.leptum
## 2023-06-08 17:09:46.371067 INFO::Creating boxplot for categorical data, dysbiosisCD vs Dorea.longicatena
## 2023-06-08 17:09:46.616264 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.caccae
## 2023-06-08 17:09:46.866061 INFO::Creating boxplot for categorical data, dysbiosisCD vs Lachnospiraceae.bacterium.3.1.46FAA
## 2023-06-08 17:09:47.118453 INFO::Creating boxplot for categorical data, dysbiosisCD vs Escherichia.coli
## 2023-06-08 17:09:47.35538 INFO::Creating boxplot for categorical data, dysbiosisCD vs Klebsiella.pneumoniae
## 2023-06-08 17:09:47.597149 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bilophila.unclassified
## 2023-06-08 17:09:47.828089 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.finegoldii
## 2023-06-08 17:09:48.05924 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.eligens
## 2023-06-08 17:09:48.299189 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.stercoris
## 2023-06-08 17:09:48.546824 INFO::Creating boxplot for categorical data, dysbiosisCD vs Coprococcus.comes
## 2023-06-08 17:09:48.780621 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.fragilis
## 2023-06-08 17:09:49.086488 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.finegoldii
## 2023-06-08 17:09:49.343498 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.hallii
## 2023-06-08 17:09:49.603178 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.faecis
## 2023-06-08 17:09:49.846163 INFO::Creating boxplot for categorical data, dysbiosisCD vs Paraprevotella.clara
## 2023-06-08 17:09:50.089374 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.siraeum
## 2023-06-08 17:09:50.331439 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.merdae
## 2023-06-08 17:09:50.564939 INFO::Creating boxplot for categorical data, dysbiosisCD vs Paraprevotella.unclassified
## 2023-06-08 17:09:50.812433 INFO::Creating boxplot for categorical data, dysbiosisCD vs Collinsella.aerofaciens
## 2023-06-08 17:09:51.045819 INFO::Creating boxplot for categorical data, dysbiosisCD vs Odoribacter.splanchnicus
## 2023-06-08 17:09:51.305153 INFO::Creating boxplot for categorical data, dysbiosisCD vs Clostridium.clostridioforme
## 2023-06-08 17:09:51.55458 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.eggerthii
## 2023-06-08 17:09:51.814586 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.onderdonkii
## 2023-06-08 17:09:52.066916 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.ventriosum
## 2023-06-08 17:09:52.331789 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.lactaris
## 2023-06-08 17:09:52.589842 INFO::Creating boxplot for categorical data, dysbiosisCD vs Burkholderiales.bacterium.1.1.47
## 2023-06-08 17:09:52.823988 INFO::Creating boxplot for categorical data, dysbiosisCD vs Dialister.invisus
## 2023-06-08 17:09:53.072474 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.bromii
## 2023-06-08 17:09:53.322831 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parasutterella.excrementihominis
## 2023-06-08 17:09:53.561491 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.unclassified
## 2023-06-08 17:09:53.8077 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.torques
## 2023-06-08 17:09:54.035248 INFO::Creating boxplot for categorical data, dysbiosisCD vs Coprobacillus.unclassified
## 2023-06-08 17:09:54.286466 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.cellulosilyticus
## 2023-06-08 17:09:54.529061 INFO::Creating boxplot for categorical data, dysbiosisCD vs Roseburia.intestinalis
## 2023-06-08 17:09:54.762491 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.unclassified
## 2023-06-08 17:09:55.009547 INFO::Creating boxplot for categorical data, dysbiosisCD vs Acidaminococcus.unclassified
## 2023-06-08 17:09:55.253222 INFO::Creating boxplot for categorical data, dysbiosisCD vs Barnesiella.intestinihominis
## 2023-06-08 17:09:55.499022 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.massiliensis
## 2023-06-08 17:09:55.753119 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.sp.3.1.31
## 2023-06-08 17:09:55.996019 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.salyersiae
## 2023-06-08 17:09:56.226094 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroidales.bacterium.ph8
## 2023-06-08 17:09:56.465494 INFO::Creating boxplot for categorical data, dysbiosisCD vs Clostridium.citroniae
## 2023-06-08 17:09:56.711182 INFO::Creating boxplot for categorical data, dysbiosisCD vs Flavonifractor.plautii
## 2023-06-08 17:09:56.950181 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.goldsteinii
## 2023-06-08 17:09:57.205566 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bifidobacterium.longum
## 2023-06-08 17:09:57.484909 INFO::Creating boxplot for categorical data, dysbiosisCD vs Lachnospiraceae.bacterium.1.1.57FAA
## 2023-06-08 17:10:00.582011 INFO::Plotting data for metadata number 2, dysbiosisUC
## 2023-06-08 17:10:00.583991 INFO::Creating boxplot for categorical data, dysbiosisUC vs Subdoligranulum.unclassified
## 2023-06-08 17:10:00.819352 INFO::Creating boxplot for categorical data, dysbiosisUC vs Faecalibacterium.prausnitzii
## 2023-06-08 17:10:01.068778 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.caccae
## 2023-06-08 17:10:01.335801 INFO::Creating boxplot for categorical data, dysbiosisUC vs Oscillibacter.unclassified
## 2023-06-08 17:10:01.585276 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.uniformis
## 2023-06-08 17:10:01.847997 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.siraeum
## 2023-06-08 17:10:02.112376 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.ovatus
## 2023-06-08 17:10:02.355929 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.fragilis
## 2023-06-08 17:10:02.628998 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.shahii
## 2023-06-08 17:10:02.871145 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.rectale
## 2023-06-08 17:10:03.12234 INFO::Creating boxplot for categorical data, dysbiosisUC vs Roseburia.hominis
## 2023-06-08 17:10:03.385527 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.putredinis
## 2023-06-08 17:10:03.640373 INFO::Creating boxplot for categorical data, dysbiosisUC vs Lachnospiraceae.bacterium.3.1.46FAA
## 2023-06-08 17:10:03.912247 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.hallii
## 2023-06-08 17:10:04.170933 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.xylanisolvens
## 2023-06-08 17:10:04.41841 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.stercoris
## 2023-06-08 17:10:04.680583 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.finegoldii
## 2023-06-08 17:10:04.926243 INFO::Creating boxplot for categorical data, dysbiosisUC vs Barnesiella.intestinihominis
## 2023-06-08 17:10:05.180292 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.leptum
## 2023-06-08 17:10:05.432076 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.gnavus
## 2023-06-08 17:10:05.67175 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.onderdonkii
## 2023-06-08 17:10:05.926365 INFO::Creating boxplot for categorical data, dysbiosisUC vs Flavonifractor.plautii
## 2023-06-08 17:10:06.181548 INFO::Creating boxplot for categorical data, dysbiosisUC vs Parabacteroides.merdae
## 2023-06-08 17:10:06.424266 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.cellulosilyticus
## 2023-06-08 17:10:06.682382 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bifidobacterium.longum
## 2023-06-08 17:10:06.998253 INFO::Creating boxplot for categorical data, dysbiosisUC vs Klebsiella.pneumoniae
## 2023-06-08 17:10:07.250354 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.citroniae
## 2023-06-08 17:10:07.512235 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.ventriosum
## 2023-06-08 17:10:07.753868 INFO::Creating boxplot for categorical data, dysbiosisUC vs Parabacteroides.distasonis
## 2023-06-08 17:10:08.012712 INFO::Creating boxplot for categorical data, dysbiosisUC vs Parabacteroides.goldsteinii
## 2023-06-08 17:10:08.276797 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.torques
## 2023-06-08 17:10:08.526506 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroidales.bacterium.ph8
## 2023-06-08 17:10:08.813648 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.obeum
## 2023-06-08 17:10:09.055188 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bifidobacterium.bifidum
## 2023-06-08 17:10:09.320322 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bifidobacterium.adolescentis
## 2023-06-08 17:10:09.567887 INFO::Creating boxplot for categorical data, dysbiosisUC vs Collinsella.aerofaciens
## 2023-06-08 17:10:09.810602 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.hathewayi
## 2023-06-08 17:10:10.069259 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bilophila.unclassified
## 2023-06-08 17:10:10.316788 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.eligens
## 2023-06-08 17:10:10.576232 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.vulgatus
## 2023-06-08 17:10:10.836414 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.bolteae
## 2023-06-08 17:10:11.08049 INFO::Creating boxplot for categorical data, dysbiosisUC vs Dialister.invisus
## 2023-06-08 17:10:11.334192 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.lactaris
## 2023-06-08 17:10:11.572246 INFO::Creating boxplot for categorical data, dysbiosisUC vs Burkholderiales.bacterium.1.1.47
## 2023-06-08 17:10:14.673452 INFO::Plotting data for metadata number 3, dysbiosisnonIBD
## 2023-06-08 17:10:14.675345 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Faecalibacterium.prausnitzii
## 2023-06-08 17:10:14.900805 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.rectale
## 2023-06-08 17:10:15.152141 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.sp.3.1.31
## 2023-06-08 17:10:15.412104 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.hominis
## 2023-06-08 17:10:15.660236 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Subdoligranulum.unclassified
## 2023-06-08 17:10:15.907889 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Lachnospiraceae.bacterium.3.1.46FAA
## 2023-06-08 17:10:16.207004 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Ruminococcus.torques
## 2023-06-08 17:10:16.454122 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Clostridium.leptum
## 2023-06-08 17:10:16.725617 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Odoribacter.splanchnicus
## 2023-06-08 17:10:16.990898 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Escherichia.coli
## 2023-06-08 17:10:17.23692 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Ruminococcus.obeum
## 2023-06-08 17:10:17.50265 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bilophila.unclassified
## 2023-06-08 17:10:17.754826 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.uniformis
## 2023-06-08 17:10:18.020862 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.eligens
## 2023-06-08 17:10:18.291238 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Klebsiella.pneumoniae
## 2023-06-08 17:10:18.543018 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Ruminococcus.bromii
## 2023-06-08 17:10:18.800429 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.vulgatus
## 2023-06-08 17:10:19.042431 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs C2likevirus.unclassified
## 2023-06-08 17:10:19.32513 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bifidobacterium.adolescentis
## 2023-06-08 17:10:19.604536 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Alistipes.finegoldii
## 2023-06-08 17:10:19.848318 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.inulinivorans
## 2023-06-08 17:10:20.114906 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Alistipes.onderdonkii
## 2023-06-08 17:10:20.385208 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.unclassified
## 2023-06-08 17:10:20.642366 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.hallii
## 2023-06-08 17:10:20.910843 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.intestinalis
## 2023-06-08 17:10:21.173732 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Prevotella.copri
## 2023-06-08 17:10:21.42482 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.dorei
## 2023-06-08 17:10:21.681371 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.fragilis
## 2023-06-08 17:10:21.939996 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Alistipes.shahii
## 2023-06-08 17:10:25.033761 INFO::Plotting data for metadata number 4, antibiotics
## 2023-06-08 17:10:25.035509 INFO::Creating boxplot for categorical data, antibiotics vs Roseburia.inulinivorans
## 2023-06-08 17:10:25.331712 INFO::Creating boxplot for categorical data, antibiotics vs Roseburia.hominis
## 2023-06-08 17:10:25.577457 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.rectale
## 2023-06-08 17:10:25.837709 INFO::Creating boxplot for categorical data, antibiotics vs Dialister.invisus
## 2023-06-08 17:10:26.094591 INFO::Creating boxplot for categorical data, antibiotics vs Roseburia.intestinalis
## 2023-06-08 17:10:26.340087 INFO::Creating boxplot for categorical data, antibiotics vs Dorea.longicatena
## 2023-06-08 17:10:26.602204 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.callidus
## 2023-06-08 17:10:26.839547 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.bromii
## 2023-06-08 17:10:27.087876 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.obeum
## 2023-06-08 17:10:27.322195 INFO::Creating boxplot for categorical data, antibiotics vs Klebsiella.pneumoniae
## 2023-06-08 17:10:27.587285 INFO::Creating boxplot for categorical data, antibiotics vs Bifidobacterium.adolescentis
## 2023-06-08 17:10:27.826503 INFO::Creating boxplot for categorical data, antibiotics vs Faecalibacterium.prausnitzii
## 2023-06-08 17:10:28.069338 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.hallii
## 2023-06-08 17:10:28.335789 INFO::Creating boxplot for categorical data, antibiotics vs Bilophila.unclassified
## 2023-06-08 17:10:28.571266 INFO::Creating boxplot for categorical data, antibiotics vs Clostridium.leptum
## 2023-06-08 17:10:28.827296 INFO::Creating boxplot for categorical data, antibiotics vs Lachnospiraceae.bacterium.3.1.46FAA
## 2023-06-08 17:10:29.063914 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.finegoldii
## 2023-06-08 17:10:29.321934 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.sp.3.1.31
## 2023-06-08 17:10:29.573022 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.fragilis
## 2023-06-08 17:10:29.804008 INFO::Creating boxplot for categorical data, antibiotics vs Alistipes.onderdonkii
## 2023-06-08 17:10:30.05745 INFO::Creating boxplot for categorical data, antibiotics vs Sutterella.wadsworthensis
## 2023-06-08 17:10:30.291751 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.eligens
## 2023-06-08 17:10:30.542802 INFO::Creating boxplot for categorical data, antibiotics vs Collinsella.aerofaciens
## 2023-06-08 17:10:30.79821 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.thetaiotaomicron
## 2023-06-08 17:10:31.034576 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.eggerthii
## 2023-06-08 17:10:31.295001 INFO::Creating boxplot for categorical data, antibiotics vs Haemophilus.parainfluenzae
## 2023-06-08 17:10:31.541128 INFO::Creating boxplot for categorical data, antibiotics vs Bifidobacterium.pseudocatenulatum
## 2023-06-08 17:10:31.779526 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.torques
## 2023-06-08 17:10:32.031927 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.ventriosum
## 2023-06-08 17:10:32.312027 INFO::Creating boxplot for categorical data, antibiotics vs Parasutterella.excrementihominis
## 2023-06-08 17:10:32.559823 INFO::Creating boxplot for categorical data, antibiotics vs Peptostreptococcaceae.noname.unclassified
## 2023-06-08 17:10:32.818698 INFO::Creating boxplot for categorical data, antibiotics vs Veillonella.dispar
## 2023-06-08 17:10:33.056541 INFO::Creating boxplot for categorical data, antibiotics vs Veillonella.atypica
## 2023-06-08 17:10:33.295286 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroidales.bacterium.ph8
## 2023-06-08 17:10:33.545663 INFO::Creating boxplot for categorical data, antibiotics vs Clostridium.nexile
## 2023-06-08 17:10:34.06018 INFO::Creating boxplot for categorical data, antibiotics vs Burkholderiales.bacterium.1.1.47
## 2023-06-08 17:10:34.270846 INFO::Creating boxplot for categorical data, antibiotics vs Lachnospiraceae.bacterium.1.1.57FAA
## 2023-06-08 17:10:34.495212 INFO::Creating boxplot for categorical data, antibiotics vs Akkermansia.muciniphila
## 2023-06-08 17:10:34.713989 INFO::Creating boxplot for categorical data, antibiotics vs Clostridium.citroniae
## 2023-06-08 17:10:34.940975 INFO::Creating boxplot for categorical data, antibiotics vs Odoribacter.splanchnicus
## 2023-06-08 17:10:37.693829 INFO::Plotting data for metadata number 5, age
## 2023-06-08 17:10:37.695557 INFO::Creating scatter plot for continuous data, age vs Haemophilus.parainfluenzae
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-06-08 17:10:37.969122 INFO::Creating scatter plot for continuous data, age vs Bifidobacterium.pseudocatenulatum
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-06-08 17:10:38.27937 INFO::Creating scatter plot for continuous data, age vs Faecalibacterium.prausnitzii
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-06-08 17:10:38.515707 INFO::Creating scatter plot for continuous data, age vs Clostridium.clostridioforme
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-06-08 17:10:38.769812 INFO::Creating scatter plot for continuous data, age vs Veillonella.parvula
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-06-08 17:10:39.011322 INFO::Creating scatter plot for continuous data, age vs Subdoligranulum.unclassified
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-06-08 17:10:39.242856 INFO::Creating scatter plot for continuous data, age vs Clostridium.symbiosum
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-06-08 17:10:39.522103 INFO::Creating scatter plot for continuous data, age vs Ruminococcus.gnavus
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-06-08 17:10:39.76654 INFO::Creating scatter plot for continuous data, age vs Dialister.invisus
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-06-08 17:10:40.007882 INFO::Creating scatter plot for continuous data, age vs Veillonella.dispar
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-06-08 17:10:40.24437 INFO::Creating scatter plot for continuous data, age vs Veillonella.unclassified
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-06-08 17:10:40.485373 INFO::Creating scatter plot for continuous data, age vs Bacteroides.thetaiotaomicron
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-06-08 17:10:40.725618 INFO::Creating scatter plot for continuous data, age vs Ruminococcus.bromii
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-06-08 17:10:40.976024 INFO::Creating scatter plot for continuous data, age vs Bacteroides.intestinalis
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-06-08 17:10:41.206684 INFO::Creating scatter plot for continuous data, age vs Eubacterium.siraeum
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-06-08 17:10:41.447055 INFO::Creating scatter plot for continuous data, age vs Prevotella.copri
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-06-08 17:10:41.696936 INFO::Creating scatter plot for continuous data, age vs Alistipes.unclassified
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-06-08 17:10:41.935344 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 (`geom_point()`).
## 2023-06-08 17:10:42.21984 INFO::Creating scatter plot for continuous data, age vs Bifidobacterium.longum
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-06-08 17:10:42.457673 INFO::Creating scatter plot for continuous data, age vs Akkermansia.muciniphila
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-06-08 17:10:42.712127 INFO::Creating scatter plot for continuous data, age vs Collinsella.aerofaciens
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-06-08 17:10:42.956886 INFO::Creating scatter plot for continuous data, age vs Parabacteroides.distasonis
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-06-08 17:10:45.986702 INFO::Plotting data for metadata number 6, diagnosis
## 2023-06-08 17:10:45.988495 INFO::Creating boxplot for categorical data, diagnosis vs Bifidobacterium.adolescentis
## 2023-06-08 17:10:46.207627 INFO::Creating boxplot for categorical data, diagnosis vs Akkermansia.muciniphila
## 2023-06-08 17:10:46.452422 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.bolteae
## 2023-06-08 17:10:46.684458 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.putredinis
## 2023-06-08 17:10:46.936952 INFO::Creating boxplot for categorical data, diagnosis vs Coprobacillus.unclassified
## 2023-06-08 17:10:47.184884 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.clostridioforme
## 2023-06-08 17:10:47.417888 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.shahii
## 2023-06-08 17:10:47.66517 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.bromii
## 2023-06-08 17:10:47.943607 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.leptum
## 2023-06-08 17:10:48.181371 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.symbiosum
## 2023-06-08 17:10:48.430248 INFO::Creating boxplot for categorical data, diagnosis vs Roseburia.inulinivorans
## 2023-06-08 17:10:48.680526 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.bromii
## 2023-06-08 17:10:48.92239 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.shahii
## 2023-06-08 17:10:49.169079 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.finegoldii
## 2023-06-08 17:10:49.417779 INFO::Creating boxplot for categorical data, diagnosis vs Parabacteroides.goldsteinii
## 2023-06-08 17:10:49.653475 INFO::Creating boxplot for categorical data, diagnosis vs Bilophila.unclassified
## 2023-06-08 17:10:49.897609 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.leptum
## 2023-06-08 17:10:50.147992 INFO::Creating boxplot for categorical data, diagnosis vs Roseburia.hominis
## 2023-06-08 17:10:50.388274 INFO::Creating boxplot for categorical data, diagnosis vs Coprobacillus.unclassified
## 2023-06-08 17:10:50.683982 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.onderdonkii
## 2023-06-08 17:10:50.936483 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.ventriosum
## 2023-06-08 17:10:51.176794 INFO::Creating boxplot for categorical data, diagnosis vs Roseburia.hominis
## 2023-06-08 17:10:51.429697 INFO::Creating boxplot for categorical data, diagnosis vs Sutterella.wadsworthensis
## 2023-06-08 17:10:51.680815 INFO::Creating boxplot for categorical data, diagnosis vs Akkermansia.muciniphila
## 2023-06-08 17:10:51.919346 INFO::Creating boxplot for categorical data, diagnosis vs Sutterella.wadsworthensis
## 2023-06-08 17:10:52.171899 INFO::Creating boxplot for categorical data, diagnosis vs Subdoligranulum.unclassified
## 2023-06-08 17:10:52.405972 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.symbiosum
## 2023-06-08 17:10:52.66466 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.onderdonkii
## 2023-06-08 17:10:52.919895 INFO::Creating boxplot for categorical data, diagnosis vs Parabacteroides.unclassified
## 2023-06-08 17:10:53.157689 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.lactaris
## 2023-06-08 17:10:53.412192 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.callidus
## 2023-06-08 17:10:53.661397 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.gnavus
## 2023-06-08 17:10:53.895861 INFO::Creating boxplot for categorical data, diagnosis vs Bacteroides.fragilis
## 2023-06-08 17:10:54.155168 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.rectale
## 2023-06-08 17:10:54.411168 INFO::Creating boxplot for categorical data, diagnosis vs Odoribacter.splanchnicus
## 2023-06-08 17:10:54.652509 INFO::Creating boxplot for categorical data, diagnosis vs Parabacteroides.distasonis
## 2023-06-08 17:10:54.906312 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.rectale
## 2023-06-08 17:10:55.160591 INFO::Creating boxplot for categorical data, diagnosis vs Bacteroides.finegoldii
## 2023-06-08 17:10:55.401166 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.callidus
## 2023-06-08 17:10:55.655552 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.finegoldii
## 2023-06-08 17:10:55.907137 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.putredinis
## 2023-06-08 17:10:56.143245 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.bolteae
## 2023-06-08 17:10:56.403189 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 4.3.0 RC (2023-04-13 r84269)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 22.04.2 LTS
## 
## Matrix products: default
## BLAS:   /home/biocbuild/bbs-3.17-bioc/R/lib/libRblas.so 
## LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.10.0
## 
## locale:
##  [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
##  [3] LC_TIME=en_GB              LC_COLLATE=C              
##  [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
##  [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
##  [9] LC_ADDRESS=C               LC_TELEPHONE=C            
## [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       
## 
## time zone: America/New_York
## tzcode source: system (glibc)
## 
## attached base packages:
## [1] stats     graphics  grDevices utils     datasets  methods   base     
## 
## other attached packages:
## [1] Maaslin2_1.14.1
## 
## loaded via a namespace (and not attached):
##  [1] gtable_0.3.3        biglm_0.9-2.1       xfun_0.39          
##  [4] bslib_0.4.2         ggplot2_3.4.2       lattice_0.21-8     
##  [7] numDeriv_2016.8-1.1 vctrs_0.6.2         tools_4.3.0        
## [10] generics_0.1.3      parallel_4.3.0      getopt_1.20.3      
## [13] tibble_3.2.1        fansi_1.0.4         DEoptimR_1.0-13    
## [16] cluster_2.1.4       pkgconfig_2.0.3     logging_0.10-108   
## [19] pheatmap_1.0.12     Matrix_1.5-4.1      data.table_1.14.8  
## [22] RColorBrewer_1.1-3  lifecycle_1.0.3     farver_2.1.1       
## [25] compiler_4.3.0      munsell_0.5.0       lmerTest_3.1-3     
## [28] permute_0.9-7       htmltools_0.5.5     sass_0.4.6         
## [31] hash_2.2.6.2        yaml_2.3.7          pillar_1.9.0       
## [34] nloptr_2.0.3        crayon_1.5.2        jquerylib_0.1.4    
## [37] MASS_7.3-60         cachem_1.0.8        vegan_2.6-4        
## [40] boot_1.3-28.1       nlme_3.1-162        robustbase_0.95-1  
## [43] tidyselect_1.2.0    digest_0.6.31       mvtnorm_1.2-2      
## [46] dplyr_1.1.2         labeling_0.4.2      splines_4.3.0      
## [49] pcaPP_2.0-3         fastmap_1.1.1       grid_4.3.0         
## [52] colorspace_2.1-0    cli_3.6.1           magrittr_2.0.3     
## [55] utf8_1.2.3          withr_2.5.0         scales_1.2.1       
## [58] rmarkdown_2.22      lme4_1.1-33         pbapply_1.7-0      
## [61] evaluate_0.21       knitr_1.43          mgcv_1.8-42        
## [64] rlang_1.1.1         Rcpp_1.0.10         glue_1.6.2         
## [67] optparse_1.7.3      DBI_1.1.3           minqa_1.2.5        
## [70] jsonlite_1.8.5      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.