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"
## 2024-05-01 20:05:29.223916 INFO::Writing function arguments to log file
## 2024-05-01 20:05:29.293886 INFO::Verifying options selected are valid
## 2024-05-01 20:05:29.384291 INFO::Determining format of input files
## 2024-05-01 20:05:29.387072 INFO::Input format is data samples as rows and metadata samples as rows
## 2024-05-01 20:05:29.400381 INFO::Formula for random effects: expr ~ (1 | site) + (1 | subject)
## 2024-05-01 20:05:29.403229 INFO::Formula for fixed effects: expr ~  diagnosis + dysbiosisnonIBD + dysbiosisUC + dysbiosisCD + antibiotics + age
## 2024-05-01 20:05:29.40891 INFO::Filter data based on min abundance and min prevalence
## 2024-05-01 20:05:29.411114 INFO::Total samples in data: 1595
## 2024-05-01 20:05:29.413427 INFO::Min samples required with min abundance for a feature not to be filtered: 159.500000
## 2024-05-01 20:05:29.426761 INFO::Total filtered features: 0
## 2024-05-01 20:05:29.429111 INFO::Filtered feature names from abundance and prevalence filtering:
## 2024-05-01 20:05:29.448948 INFO::Total filtered features with variance filtering: 0
## 2024-05-01 20:05:29.451008 INFO::Filtered feature names from variance filtering:
## 2024-05-01 20:05:29.452703 INFO::Running selected normalization method: TSS
## 2024-05-01 20:05:31.820614 INFO::Bypass z-score application to metadata
## 2024-05-01 20:05:31.822929 INFO::Running selected transform method: LOG
## 2024-05-01 20:05:31.867292 INFO::Running selected analysis method: LM
## 2024-05-01 20:05:32.68132 INFO::Fitting model to feature number 1, Bifidobacterium.adolescentis
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:33.383329 INFO::Fitting model to feature number 2, Bifidobacterium.bifidum
## 2024-05-01 20:05:33.730884 INFO::Fitting model to feature number 3, Bifidobacterium.longum
## 2024-05-01 20:05:34.14337 INFO::Fitting model to feature number 4, Bifidobacterium.pseudocatenulatum
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:34.675089 INFO::Fitting model to feature number 5, Collinsella.aerofaciens
## 2024-05-01 20:05:34.983997 INFO::Fitting model to feature number 6, Bacteroides.caccae
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:35.280943 INFO::Fitting model to feature number 7, Bacteroides.cellulosilyticus
## 2024-05-01 20:05:35.621178 INFO::Fitting model to feature number 8, Bacteroides.dorei
## 2024-05-01 20:05:35.990115 INFO::Fitting model to feature number 9, Bacteroides.eggerthii
## 2024-05-01 20:05:36.300704 INFO::Fitting model to feature number 10, Bacteroides.faecis
## 2024-05-01 20:05:36.621888 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
## 2024-05-01 20:05:36.883188 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
## 2024-05-01 20:05:37.206772 INFO::Fitting model to feature number 12, Bacteroides.fragilis
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:37.504817 INFO::Fitting model to feature number 13, Bacteroides.intestinalis
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:37.821378 INFO::Fitting model to feature number 14, Bacteroides.massiliensis
## 2024-05-01 20:05:38.132247 INFO::Fitting model to feature number 15, Bacteroides.ovatus
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:38.438101 INFO::Fitting model to feature number 16, Bacteroides.salyersiae
## 2024-05-01 20:05:38.757298 INFO::Fitting model to feature number 17, Bacteroides.stercoris
## 2024-05-01 20:05:39.065847 INFO::Fitting model to feature number 18, Bacteroides.thetaiotaomicron
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:39.379409 INFO::Fitting model to feature number 19, Bacteroides.uniformis
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:39.675029 INFO::Fitting model to feature number 20, Bacteroides.vulgatus
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:39.976805 INFO::Fitting model to feature number 21, Bacteroides.xylanisolvens
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:40.292266 INFO::Fitting model to feature number 22, Bacteroidales.bacterium.ph8
## 2024-05-01 20:05:40.588286 INFO::Fitting model to feature number 23, Barnesiella.intestinihominis
## 2024-05-01 20:05:40.904132 INFO::Fitting model to feature number 24, Coprobacter.fastidiosus
## 2024-05-01 20:05:41.219956 INFO::Fitting model to feature number 25, Odoribacter.splanchnicus
## 2024-05-01 20:05:41.523717 INFO::Fitting model to feature number 26, Parabacteroides.distasonis
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:41.818081 INFO::Fitting model to feature number 27, Parabacteroides.goldsteinii
## 2024-05-01 20:05:42.125741 INFO::Fitting model to feature number 28, Parabacteroides.merdae
## 2024-05-01 20:05:42.45383 INFO::Fitting model to feature number 29, Parabacteroides.unclassified
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:42.751863 INFO::Fitting model to feature number 30, Paraprevotella.clara
## 2024-05-01 20:05:43.067989 INFO::Fitting model to feature number 31, Paraprevotella.unclassified
## 2024-05-01 20:05:43.385264 INFO::Fitting model to feature number 32, Prevotella.copri
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:43.688207 INFO::Fitting model to feature number 33, Alistipes.finegoldii
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:43.988601 INFO::Fitting model to feature number 34, Alistipes.onderdonkii
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:44.333363 INFO::Fitting model to feature number 35, Alistipes.putredinis
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:44.645105 INFO::Fitting model to feature number 36, Alistipes.shahii
## 2024-05-01 20:05:44.966472 INFO::Fitting model to feature number 37, Alistipes.unclassified
## 2024-05-01 20:05:45.278872 INFO::Fitting model to feature number 38, Streptococcus.salivarius
## 2024-05-01 20:05:45.599868 INFO::Fitting model to feature number 39, Clostridium.bolteae
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:45.92379 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
## 2024-05-01 20:05:46.192524 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
## 2024-05-01 20:05:46.49507 INFO::Fitting model to feature number 41, Clostridium.clostridioforme
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:46.804759 INFO::Fitting model to feature number 42, Clostridium.hathewayi
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:47.124477 INFO::Fitting model to feature number 43, Clostridium.leptum
## 2024-05-01 20:05:47.479185 INFO::Fitting model to feature number 44, Clostridium.nexile
## 2024-05-01 20:05:47.790944 INFO::Fitting model to feature number 45, Clostridium.symbiosum
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:48.106516 INFO::Fitting model to feature number 46, Flavonifractor.plautii
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:48.419756 INFO::Fitting model to feature number 47, Eubacterium.eligens
## 2024-05-01 20:05:48.733074 INFO::Fitting model to feature number 48, Eubacterium.hallii
## 2024-05-01 20:05:49.060873 INFO::Fitting model to feature number 49, Eubacterium.rectale
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:49.362538 INFO::Fitting model to feature number 50, Eubacterium.siraeum
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:49.668286 INFO::Fitting model to feature number 51, Eubacterium.sp.3.1.31
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:49.983543 INFO::Fitting model to feature number 52, Eubacterium.ventriosum
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:50.304388 INFO::Fitting model to feature number 53, Ruminococcus.gnavus
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:50.633565 INFO::Fitting model to feature number 54, Ruminococcus.obeum
## 2024-05-01 20:05:50.9414 INFO::Fitting model to feature number 55, Ruminococcus.torques
## 2024-05-01 20:05:51.262959 INFO::Fitting model to feature number 56, Coprococcus.comes
## 2024-05-01 20:05:51.590442 INFO::Fitting model to feature number 57, Dorea.longicatena
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:52.165629 INFO::Fitting model to feature number 58, Lachnospiraceae.bacterium.1.1.57FAA
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:52.50642 INFO::Fitting model to feature number 59, Lachnospiraceae.bacterium.3.1.46FAA
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:52.798801 INFO::Fitting model to feature number 60, Roseburia.hominis
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:53.112687 INFO::Fitting model to feature number 61, Roseburia.intestinalis
## 2024-05-01 20:05:53.402899 INFO::Fitting model to feature number 62, Roseburia.inulinivorans
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:53.701709 INFO::Fitting model to feature number 63, Roseburia.unclassified
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:53.985834 INFO::Fitting model to feature number 64, Oscillibacter.unclassified
## 2024-05-01 20:05:54.314017 INFO::Fitting model to feature number 65, Peptostreptococcaceae.noname.unclassified
## 2024-05-01 20:05:54.609883 INFO::Fitting model to feature number 66, Faecalibacterium.prausnitzii
## 2024-05-01 20:05:54.951469 INFO::Fitting model to feature number 67, Ruminococcus.bromii
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:55.223923 INFO::Fitting model to feature number 68, Ruminococcus.callidus
## 2024-05-01 20:05:55.514849 INFO::Fitting model to feature number 69, Ruminococcus.lactaris
## 2024-05-01 20:05:55.832143 INFO::Fitting model to feature number 70, Subdoligranulum.unclassified
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:56.170845 INFO::Fitting model to feature number 71, Coprobacillus.unclassified
## 2024-05-01 20:05:56.463047 INFO::Fitting model to feature number 72, Acidaminococcus.unclassified
## 2024-05-01 20:05:56.882341 INFO::Fitting model to feature number 73, Dialister.invisus
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:57.190107 INFO::Fitting model to feature number 74, Veillonella.atypica
## 2024-05-01 20:05:57.49138 INFO::Fitting model to feature number 75, Veillonella.dispar
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:57.785288 INFO::Fitting model to feature number 76, Veillonella.parvula
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:05:58.103851 INFO::Fitting model to feature number 77, Veillonella.unclassified
## 2024-05-01 20:05:58.36093 INFO::Fitting model to feature number 78, Burkholderiales.bacterium.1.1.47
## 2024-05-01 20:05:58.688108 INFO::Fitting model to feature number 79, Parasutterella.excrementihominis
## 2024-05-01 20:05:59.247248 INFO::Fitting model to feature number 80, Sutterella.wadsworthensis
## 2024-05-01 20:05:59.550421 INFO::Fitting model to feature number 81, Bilophila.unclassified
## 2024-05-01 20:05:59.886897 INFO::Fitting model to feature number 82, Escherichia.coli
## 2024-05-01 20:06:00.201673 INFO::Fitting model to feature number 83, Escherichia.unclassified
## 2024-05-01 20:06:00.486859 INFO::Fitting model to feature number 84, Klebsiella.pneumoniae
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:06:00.777241 INFO::Fitting model to feature number 85, Haemophilus.parainfluenzae
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 20:06:01.087833 INFO::Fitting model to feature number 86, Akkermansia.muciniphila
## 2024-05-01 20:06:01.391291 INFO::Fitting model to feature number 87, C2likevirus.unclassified
## 2024-05-01 20:06:01.81056 INFO::Counting total values for each feature
## 2024-05-01 20:06:01.914798 INFO::Writing filtered data to file demo_output/features/filtered_data.tsv
## 2024-05-01 20:06:02.203789 INFO::Writing filtered, normalized data to file demo_output/features/filtered_data_norm.tsv
## 2024-05-01 20:06:02.551046 INFO::Writing filtered, normalized, transformed data to file demo_output/features/filtered_data_norm_transformed.tsv
## 2024-05-01 20:06:03.007852 INFO::Writing residuals to file demo_output/fits/residuals.rds
## 2024-05-01 20:06:03.110472 INFO::Writing fitted values to file demo_output/fits/fitted.rds
## 2024-05-01 20:06:03.169419 INFO::Writing extracted random effects to file demo_output/fits/ranef.rds
## 2024-05-01 20:06:03.181435 INFO::Writing all results to file (ordered by increasing q-values): demo_output/all_results.tsv
## 2024-05-01 20:06:03.198374 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
## 2024-05-01 20:06:03.20551 INFO::Writing heatmap of significant results to file: demo_output/heatmap.pdf
## 2024-05-01 20:06:03.557302 INFO::Writing association plots (one for each significant association) to output folder: demo_output
## 2024-05-01 20:06:03.569747 INFO::Plotting associations from most to least significant, grouped by metadata
## 2024-05-01 20:06:03.572214 INFO::Plotting data for metadata number 1, dysbiosisCD
## 2024-05-01 20:06:03.575855 INFO::Creating boxplot for categorical data, dysbiosisCD vs Faecalibacterium.prausnitzii
## 2024-05-01 20:06:04.424568 INFO::Creating boxplot for categorical data, dysbiosisCD vs Subdoligranulum.unclassified
## 2024-05-01 20:06:05.153441 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.uniformis
## 2024-05-01 20:06:05.757315 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.rectale
## 2024-05-01 20:06:06.352939 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.vulgatus
## 2024-05-01 20:06:06.939617 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.ovatus
## 2024-05-01 20:06:07.583848 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.obeum
## 2024-05-01 20:06:08.189071 INFO::Creating boxplot for categorical data, dysbiosisCD vs Oscillibacter.unclassified
## 2024-05-01 20:06:08.800763 INFO::Creating boxplot for categorical data, dysbiosisCD vs Roseburia.inulinivorans
## 2024-05-01 20:06:09.416359 INFO::Creating boxplot for categorical data, dysbiosisCD vs Roseburia.hominis
## 2024-05-01 20:06:10.03303 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.thetaiotaomicron
## 2024-05-01 20:06:10.618977 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.putredinis
## 2024-05-01 20:06:11.232277 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.distasonis
## 2024-05-01 20:06:11.949204 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.dorei
## 2024-05-01 20:06:12.592324 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.shahii
## 2024-05-01 20:06:13.22087 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.xylanisolvens
## 2024-05-01 20:06:13.83884 INFO::Creating boxplot for categorical data, dysbiosisCD vs Clostridium.leptum
## 2024-05-01 20:06:14.427169 INFO::Creating boxplot for categorical data, dysbiosisCD vs Dorea.longicatena
## 2024-05-01 20:06:15.063279 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.caccae
## 2024-05-01 20:06:15.647462 INFO::Creating boxplot for categorical data, dysbiosisCD vs Lachnospiraceae.bacterium.3.1.46FAA
## 2024-05-01 20:06:16.256799 INFO::Creating boxplot for categorical data, dysbiosisCD vs Escherichia.coli
## 2024-05-01 20:06:16.853709 INFO::Creating boxplot for categorical data, dysbiosisCD vs Klebsiella.pneumoniae
## 2024-05-01 20:06:17.466376 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bilophila.unclassified
## 2024-05-01 20:06:18.042813 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.finegoldii
## 2024-05-01 20:06:18.665501 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.eligens
## 2024-05-01 20:06:19.304054 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.stercoris
## 2024-05-01 20:06:19.972975 INFO::Creating boxplot for categorical data, dysbiosisCD vs Coprococcus.comes
## 2024-05-01 20:06:20.587007 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.fragilis
## 2024-05-01 20:06:21.204181 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.finegoldii
## 2024-05-01 20:06:21.791009 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.hallii
## 2024-05-01 20:06:22.411762 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.faecis
## 2024-05-01 20:06:23.015578 INFO::Creating boxplot for categorical data, dysbiosisCD vs Paraprevotella.clara
## 2024-05-01 20:06:23.625995 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.siraeum
## 2024-05-01 20:06:24.282878 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.merdae
## 2024-05-01 20:06:24.895491 INFO::Creating boxplot for categorical data, dysbiosisCD vs Paraprevotella.unclassified
## 2024-05-01 20:06:25.50814 INFO::Creating boxplot for categorical data, dysbiosisCD vs Collinsella.aerofaciens
## 2024-05-01 20:06:26.141565 INFO::Creating boxplot for categorical data, dysbiosisCD vs Odoribacter.splanchnicus
## 2024-05-01 20:06:26.671749 INFO::Creating boxplot for categorical data, dysbiosisCD vs Clostridium.clostridioforme
## 2024-05-01 20:06:27.331449 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.eggerthii
## 2024-05-01 20:06:27.955245 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.onderdonkii
## 2024-05-01 20:06:28.57827 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.ventriosum
## 2024-05-01 20:06:29.187751 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.lactaris
## 2024-05-01 20:06:29.832847 INFO::Creating boxplot for categorical data, dysbiosisCD vs Burkholderiales.bacterium.1.1.47
## 2024-05-01 20:06:30.426278 INFO::Creating boxplot for categorical data, dysbiosisCD vs Dialister.invisus
## 2024-05-01 20:06:31.066701 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.bromii
## 2024-05-01 20:06:31.685926 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parasutterella.excrementihominis
## 2024-05-01 20:06:32.294594 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.unclassified
## 2024-05-01 20:06:32.902389 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.torques
## 2024-05-01 20:06:33.49827 INFO::Creating boxplot for categorical data, dysbiosisCD vs Coprobacillus.unclassified
## 2024-05-01 20:06:34.105746 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.cellulosilyticus
## 2024-05-01 20:06:34.681564 INFO::Creating boxplot for categorical data, dysbiosisCD vs Roseburia.intestinalis
## 2024-05-01 20:06:35.255837 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.unclassified
## 2024-05-01 20:06:35.842436 INFO::Creating boxplot for categorical data, dysbiosisCD vs Acidaminococcus.unclassified
## 2024-05-01 20:06:36.440599 INFO::Creating boxplot for categorical data, dysbiosisCD vs Barnesiella.intestinihominis
## 2024-05-01 20:06:37.022462 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.massiliensis
## 2024-05-01 20:06:37.583689 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.sp.3.1.31
## 2024-05-01 20:06:38.181694 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.salyersiae
## 2024-05-01 20:06:38.775727 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroidales.bacterium.ph8
## 2024-05-01 20:06:39.334667 INFO::Creating boxplot for categorical data, dysbiosisCD vs Clostridium.citroniae
## 2024-05-01 20:06:39.913449 INFO::Creating boxplot for categorical data, dysbiosisCD vs Flavonifractor.plautii
## 2024-05-01 20:06:40.499413 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.goldsteinii
## 2024-05-01 20:06:41.098676 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bifidobacterium.longum
## 2024-05-01 20:06:41.671851 INFO::Creating boxplot for categorical data, dysbiosisCD vs Lachnospiraceae.bacterium.1.1.57FAA
## 2024-05-01 20:06:48.183826 INFO::Plotting data for metadata number 2, dysbiosisUC
## 2024-05-01 20:06:48.188323 INFO::Creating boxplot for categorical data, dysbiosisUC vs Subdoligranulum.unclassified
## 2024-05-01 20:06:48.710149 INFO::Creating boxplot for categorical data, dysbiosisUC vs Faecalibacterium.prausnitzii
## 2024-05-01 20:06:49.334238 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.caccae
## 2024-05-01 20:06:49.912979 INFO::Creating boxplot for categorical data, dysbiosisUC vs Oscillibacter.unclassified
## 2024-05-01 20:06:50.561784 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.uniformis
## 2024-05-01 20:06:51.17928 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.siraeum
## 2024-05-01 20:06:51.832623 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.ovatus
## 2024-05-01 20:06:52.411553 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.fragilis
## 2024-05-01 20:06:53.026912 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.shahii
## 2024-05-01 20:06:53.625081 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.rectale
## 2024-05-01 20:06:54.225156 INFO::Creating boxplot for categorical data, dysbiosisUC vs Roseburia.hominis
## 2024-05-01 20:06:54.856918 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.putredinis
## 2024-05-01 20:06:55.464575 INFO::Creating boxplot for categorical data, dysbiosisUC vs Lachnospiraceae.bacterium.3.1.46FAA
## 2024-05-01 20:06:56.048695 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.hallii
## 2024-05-01 20:06:56.658403 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.xylanisolvens
## 2024-05-01 20:06:57.243366 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.stercoris
## 2024-05-01 20:06:57.878375 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.finegoldii
## 2024-05-01 20:06:58.468297 INFO::Creating boxplot for categorical data, dysbiosisUC vs Barnesiella.intestinihominis
## 2024-05-01 20:06:59.107075 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.leptum
## 2024-05-01 20:06:59.713625 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.gnavus
## 2024-05-01 20:07:00.332962 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.onderdonkii
## 2024-05-01 20:07:00.941143 INFO::Creating boxplot for categorical data, dysbiosisUC vs Flavonifractor.plautii
## 2024-05-01 20:07:01.549574 INFO::Creating boxplot for categorical data, dysbiosisUC vs Parabacteroides.merdae
## 2024-05-01 20:07:02.138026 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.cellulosilyticus
## 2024-05-01 20:07:02.852372 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bifidobacterium.longum
## 2024-05-01 20:07:03.56593 INFO::Creating boxplot for categorical data, dysbiosisUC vs Klebsiella.pneumoniae
## 2024-05-01 20:07:04.185321 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.citroniae
## 2024-05-01 20:07:04.800055 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.ventriosum
## 2024-05-01 20:07:05.431782 INFO::Creating boxplot for categorical data, dysbiosisUC vs Parabacteroides.distasonis
## 2024-05-01 20:07:06.030861 INFO::Creating boxplot for categorical data, dysbiosisUC vs Parabacteroides.goldsteinii
## 2024-05-01 20:07:06.643668 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.torques
## 2024-05-01 20:07:07.256514 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroidales.bacterium.ph8
## 2024-05-01 20:07:07.865967 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.obeum
## 2024-05-01 20:07:08.501399 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bifidobacterium.bifidum
## 2024-05-01 20:07:09.111815 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bifidobacterium.adolescentis
## 2024-05-01 20:07:09.713101 INFO::Creating boxplot for categorical data, dysbiosisUC vs Collinsella.aerofaciens
## 2024-05-01 20:07:10.318935 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.hathewayi
## 2024-05-01 20:07:10.923598 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bilophila.unclassified
## 2024-05-01 20:07:11.526796 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.eligens
## 2024-05-01 20:07:12.111565 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.vulgatus
## 2024-05-01 20:07:12.72275 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.bolteae
## 2024-05-01 20:07:13.320248 INFO::Creating boxplot for categorical data, dysbiosisUC vs Dialister.invisus
## 2024-05-01 20:07:14.009313 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.lactaris
## 2024-05-01 20:07:14.601911 INFO::Creating boxplot for categorical data, dysbiosisUC vs Burkholderiales.bacterium.1.1.47
## 2024-05-01 20:07:21.091051 INFO::Plotting data for metadata number 3, dysbiosisnonIBD
## 2024-05-01 20:07:21.095227 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Faecalibacterium.prausnitzii
## 2024-05-01 20:07:21.639932 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.rectale
## 2024-05-01 20:07:22.222054 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.sp.3.1.31
## 2024-05-01 20:07:22.819405 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.hominis
## 2024-05-01 20:07:23.357916 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Subdoligranulum.unclassified
## 2024-05-01 20:07:23.975344 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Lachnospiraceae.bacterium.3.1.46FAA
## 2024-05-01 20:07:24.580296 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Ruminococcus.torques
## 2024-05-01 20:07:25.222693 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Clostridium.leptum
## 2024-05-01 20:07:25.860334 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Odoribacter.splanchnicus
## 2024-05-01 20:07:26.509237 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Escherichia.coli
## 2024-05-01 20:07:27.155546 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Ruminococcus.obeum
## 2024-05-01 20:07:27.815617 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bilophila.unclassified
## 2024-05-01 20:07:28.468689 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.uniformis
## 2024-05-01 20:07:29.081394 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.eligens
## 2024-05-01 20:07:29.729034 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Klebsiella.pneumoniae
## 2024-05-01 20:07:30.341413 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Ruminococcus.bromii
## 2024-05-01 20:07:30.943685 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.vulgatus
## 2024-05-01 20:07:31.532755 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs C2likevirus.unclassified
## 2024-05-01 20:07:32.15455 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bifidobacterium.adolescentis
## 2024-05-01 20:07:32.730506 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Alistipes.finegoldii
## 2024-05-01 20:07:33.339963 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.inulinivorans
## 2024-05-01 20:07:33.915836 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Alistipes.onderdonkii
## 2024-05-01 20:07:34.520831 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.unclassified
## 2024-05-01 20:07:35.127747 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.hallii
## 2024-05-01 20:07:35.728806 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.intestinalis
## 2024-05-01 20:07:36.33802 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Prevotella.copri
## 2024-05-01 20:07:36.934701 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.dorei
## 2024-05-01 20:07:37.51954 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.fragilis
## 2024-05-01 20:07:38.209801 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Alistipes.shahii
## 2024-05-01 20:07:44.860095 INFO::Plotting data for metadata number 4, antibiotics
## 2024-05-01 20:07:44.863822 INFO::Creating boxplot for categorical data, antibiotics vs Roseburia.inulinivorans
## 2024-05-01 20:07:45.336762 INFO::Creating boxplot for categorical data, antibiotics vs Roseburia.hominis
## 2024-05-01 20:07:45.902638 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.rectale
## 2024-05-01 20:07:46.459074 INFO::Creating boxplot for categorical data, antibiotics vs Dialister.invisus
## 2024-05-01 20:07:47.031269 INFO::Creating boxplot for categorical data, antibiotics vs Roseburia.intestinalis
## 2024-05-01 20:07:47.621588 INFO::Creating boxplot for categorical data, antibiotics vs Dorea.longicatena
## 2024-05-01 20:07:48.158061 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.callidus
## 2024-05-01 20:07:48.713083 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.bromii
## 2024-05-01 20:07:49.231471 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.obeum
## 2024-05-01 20:07:49.77557 INFO::Creating boxplot for categorical data, antibiotics vs Klebsiella.pneumoniae
## 2024-05-01 20:07:50.327539 INFO::Creating boxplot for categorical data, antibiotics vs Bifidobacterium.adolescentis
## 2024-05-01 20:07:50.877317 INFO::Creating boxplot for categorical data, antibiotics vs Faecalibacterium.prausnitzii
## 2024-05-01 20:07:51.410361 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.hallii
## 2024-05-01 20:07:51.997783 INFO::Creating boxplot for categorical data, antibiotics vs Bilophila.unclassified
## 2024-05-01 20:07:52.561445 INFO::Creating boxplot for categorical data, antibiotics vs Clostridium.leptum
## 2024-05-01 20:07:53.126715 INFO::Creating boxplot for categorical data, antibiotics vs Lachnospiraceae.bacterium.3.1.46FAA
## 2024-05-01 20:07:53.717169 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.finegoldii
## 2024-05-01 20:07:54.270281 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.sp.3.1.31
## 2024-05-01 20:07:54.861835 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.fragilis
## 2024-05-01 20:07:55.40916 INFO::Creating boxplot for categorical data, antibiotics vs Alistipes.onderdonkii
## 2024-05-01 20:07:55.983743 INFO::Creating boxplot for categorical data, antibiotics vs Sutterella.wadsworthensis
## 2024-05-01 20:07:56.537825 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.eligens
## 2024-05-01 20:07:57.086651 INFO::Creating boxplot for categorical data, antibiotics vs Collinsella.aerofaciens
## 2024-05-01 20:07:57.62354 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.thetaiotaomicron
## 2024-05-01 20:07:58.190157 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.eggerthii
## 2024-05-01 20:07:58.781156 INFO::Creating boxplot for categorical data, antibiotics vs Haemophilus.parainfluenzae
## 2024-05-01 20:07:59.369002 INFO::Creating boxplot for categorical data, antibiotics vs Bifidobacterium.pseudocatenulatum
## 2024-05-01 20:07:59.926255 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.torques
## 2024-05-01 20:08:00.487309 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.ventriosum
## 2024-05-01 20:08:01.048767 INFO::Creating boxplot for categorical data, antibiotics vs Parasutterella.excrementihominis
## 2024-05-01 20:08:01.614274 INFO::Creating boxplot for categorical data, antibiotics vs Peptostreptococcaceae.noname.unclassified
## 2024-05-01 20:08:02.179001 INFO::Creating boxplot for categorical data, antibiotics vs Veillonella.dispar
## 2024-05-01 20:08:02.741821 INFO::Creating boxplot for categorical data, antibiotics vs Veillonella.atypica
## 2024-05-01 20:08:03.277045 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroidales.bacterium.ph8
## 2024-05-01 20:08:03.822593 INFO::Creating boxplot for categorical data, antibiotics vs Clostridium.nexile
## 2024-05-01 20:08:04.387472 INFO::Creating boxplot for categorical data, antibiotics vs Burkholderiales.bacterium.1.1.47
## 2024-05-01 20:08:04.941859 INFO::Creating boxplot for categorical data, antibiotics vs Lachnospiraceae.bacterium.1.1.57FAA
## 2024-05-01 20:08:05.496717 INFO::Creating boxplot for categorical data, antibiotics vs Akkermansia.muciniphila
## 2024-05-01 20:08:06.096593 INFO::Creating boxplot for categorical data, antibiotics vs Clostridium.citroniae
## 2024-05-01 20:08:06.644074 INFO::Creating boxplot for categorical data, antibiotics vs Odoribacter.splanchnicus
## 2024-05-01 20:08:12.979639 INFO::Plotting data for metadata number 5, age
## 2024-05-01 20:08:12.983991 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()`).
## 2024-05-01 20:08:13.633811 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()`).
## 2024-05-01 20:08:14.463974 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()`).
## 2024-05-01 20:08:15.028401 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()`).
## 2024-05-01 20:08:15.640338 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()`).
## 2024-05-01 20:08:16.237229 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()`).
## 2024-05-01 20:08:16.829839 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()`).
## 2024-05-01 20:08:17.422628 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()`).
## 2024-05-01 20:08:18.0424 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()`).
## 2024-05-01 20:08:18.658658 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()`).
## 2024-05-01 20:08:19.26449 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()`).
## 2024-05-01 20:08:19.867885 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()`).
## 2024-05-01 20:08:20.457841 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()`).
## 2024-05-01 20:08:21.065137 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()`).
## 2024-05-01 20:08:21.642111 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()`).
## 2024-05-01 20:08:22.236468 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()`).
## 2024-05-01 20:08:22.872772 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()`).
## 2024-05-01 20:08:23.441584 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()`).
## 2024-05-01 20:08:24.054672 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()`).
## 2024-05-01 20:08:24.646071 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()`).
## 2024-05-01 20:08:25.231803 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()`).
## 2024-05-01 20:08:25.829313 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()`).
## 2024-05-01 20:08:32.569207 INFO::Plotting data for metadata number 6, diagnosis
## 2024-05-01 20:08:32.574205 INFO::Creating boxplot for categorical data, diagnosis vs Bifidobacterium.adolescentis
## 2024-05-01 20:08:33.129346 INFO::Creating boxplot for categorical data, diagnosis vs Akkermansia.muciniphila
## 2024-05-01 20:08:33.76425 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.bolteae
## 2024-05-01 20:08:34.471063 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.putredinis
## 2024-05-01 20:08:35.118382 INFO::Creating boxplot for categorical data, diagnosis vs Coprobacillus.unclassified
## 2024-05-01 20:08:35.74679 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.clostridioforme
## 2024-05-01 20:08:36.384626 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.shahii
## 2024-05-01 20:08:37.010984 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.bromii
## 2024-05-01 20:08:37.604599 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.leptum
## 2024-05-01 20:08:38.201405 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.symbiosum
## 2024-05-01 20:08:38.85842 INFO::Creating boxplot for categorical data, diagnosis vs Roseburia.inulinivorans
## 2024-05-01 20:08:39.406656 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.bromii
## 2024-05-01 20:08:40.039745 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.shahii
## 2024-05-01 20:08:40.640396 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.finegoldii
## 2024-05-01 20:08:41.215466 INFO::Creating boxplot for categorical data, diagnosis vs Parabacteroides.goldsteinii
## 2024-05-01 20:08:41.828995 INFO::Creating boxplot for categorical data, diagnosis vs Bilophila.unclassified
## 2024-05-01 20:08:42.39588 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.leptum
## 2024-05-01 20:08:43.041829 INFO::Creating boxplot for categorical data, diagnosis vs Roseburia.hominis
## 2024-05-01 20:08:43.634238 INFO::Creating boxplot for categorical data, diagnosis vs Coprobacillus.unclassified
## 2024-05-01 20:08:44.265749 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.onderdonkii
## 2024-05-01 20:08:44.876638 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.ventriosum
## 2024-05-01 20:08:45.508647 INFO::Creating boxplot for categorical data, diagnosis vs Roseburia.hominis
## 2024-05-01 20:08:46.104072 INFO::Creating boxplot for categorical data, diagnosis vs Sutterella.wadsworthensis
## 2024-05-01 20:08:46.697831 INFO::Creating boxplot for categorical data, diagnosis vs Akkermansia.muciniphila
## 2024-05-01 20:08:47.273822 INFO::Creating boxplot for categorical data, diagnosis vs Sutterella.wadsworthensis
## 2024-05-01 20:08:47.843137 INFO::Creating boxplot for categorical data, diagnosis vs Subdoligranulum.unclassified
## 2024-05-01 20:08:48.448287 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.symbiosum
## 2024-05-01 20:08:49.031946 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.onderdonkii
## 2024-05-01 20:08:49.62576 INFO::Creating boxplot for categorical data, diagnosis vs Parabacteroides.unclassified
## 2024-05-01 20:08:50.218985 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.lactaris
## 2024-05-01 20:08:50.793859 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.callidus
## 2024-05-01 20:08:51.370045 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.gnavus
## 2024-05-01 20:08:51.933909 INFO::Creating boxplot for categorical data, diagnosis vs Bacteroides.fragilis
## 2024-05-01 20:08:52.517116 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.rectale
## 2024-05-01 20:08:53.062746 INFO::Creating boxplot for categorical data, diagnosis vs Odoribacter.splanchnicus
## 2024-05-01 20:08:53.695025 INFO::Creating boxplot for categorical data, diagnosis vs Parabacteroides.distasonis
## 2024-05-01 20:08:54.265945 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.rectale
## 2024-05-01 20:08:54.837516 INFO::Creating boxplot for categorical data, diagnosis vs Bacteroides.finegoldii
## 2024-05-01 20:08:55.460538 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.callidus
## 2024-05-01 20:08:56.010409 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.finegoldii
## 2024-05-01 20:08:56.604304 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.putredinis
## 2024-05-01 20:08:57.180607 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.bolteae
## 2024-05-01 20:08:57.779495 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 Patched (2024-04-24 r86482)
## Platform: x86_64-apple-darwin20
## Running under: macOS Monterey 12.7.4
## 
## Matrix products: default
## BLAS:   /Library/Frameworks/R.framework/Versions/4.4-x86_64/Resources/lib/libRblas.0.dylib 
## LAPACK: /Library/Frameworks/R.framework/Versions/4.4-x86_64/Resources/lib/libRlapack.dylib;  LAPACK version 3.12.0
## 
## locale:
## [1] C/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
## 
## time zone: America/New_York
## tzcode source: internal
## 
## attached base packages:
## [1] stats     graphics  grDevices utils     datasets  methods   base     
## 
## other attached packages:
## [1] Maaslin2_1.19.0
## 
## loaded via a namespace (and not attached):
##  [1] gtable_0.3.5        biglm_0.9-2.1       xfun_0.43          
##  [4] bslib_0.7.0         ggplot2_3.5.1       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.