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 19:45:28.088174 INFO::Writing function arguments to log file
## 2024-05-01 19:45:28.130269 INFO::Verifying options selected are valid
## 2024-05-01 19:45:28.168316 INFO::Determining format of input files
## 2024-05-01 19:45:28.170305 INFO::Input format is data samples as rows and metadata samples as rows
## 2024-05-01 19:45:28.180242 INFO::Formula for random effects: expr ~ (1 | site) + (1 | subject)
## 2024-05-01 19:45:28.18598 INFO::Formula for fixed effects: expr ~  diagnosis + dysbiosisnonIBD + dysbiosisUC + dysbiosisCD + antibiotics + age
## 2024-05-01 19:45:28.189857 INFO::Filter data based on min abundance and min prevalence
## 2024-05-01 19:45:28.191887 INFO::Total samples in data: 1595
## 2024-05-01 19:45:28.19368 INFO::Min samples required with min abundance for a feature not to be filtered: 159.500000
## 2024-05-01 19:45:28.199404 INFO::Total filtered features: 0
## 2024-05-01 19:45:28.20108 INFO::Filtered feature names from abundance and prevalence filtering:
## 2024-05-01 19:45:28.21058 INFO::Total filtered features with variance filtering: 0
## 2024-05-01 19:45:28.212551 INFO::Filtered feature names from variance filtering:
## 2024-05-01 19:45:28.214045 INFO::Running selected normalization method: TSS
## 2024-05-01 19:45:29.285304 INFO::Bypass z-score application to metadata
## 2024-05-01 19:45:29.287202 INFO::Running selected transform method: LOG
## 2024-05-01 19:45:29.31502 INFO::Running selected analysis method: LM
## 2024-05-01 19:45:29.638224 INFO::Fitting model to feature number 1, Bifidobacterium.adolescentis
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:30.090711 INFO::Fitting model to feature number 2, Bifidobacterium.bifidum
## 2024-05-01 19:45:30.505676 INFO::Fitting model to feature number 3, Bifidobacterium.longum
## 2024-05-01 19:45:30.740949 INFO::Fitting model to feature number 4, Bifidobacterium.pseudocatenulatum
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:30.984054 INFO::Fitting model to feature number 5, Collinsella.aerofaciens
## 2024-05-01 19:45:31.191236 INFO::Fitting model to feature number 6, Bacteroides.caccae
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:31.335677 INFO::Fitting model to feature number 7, Bacteroides.cellulosilyticus
## 2024-05-01 19:45:31.516864 INFO::Fitting model to feature number 8, Bacteroides.dorei
## 2024-05-01 19:45:31.735293 INFO::Fitting model to feature number 9, Bacteroides.eggerthii
## 2024-05-01 19:45:31.971917 INFO::Fitting model to feature number 10, Bacteroides.faecis
## 2024-05-01 19:45:32.188698 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 19:45:32.356816 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 19:45:32.573364 INFO::Fitting model to feature number 12, Bacteroides.fragilis
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:32.793931 INFO::Fitting model to feature number 13, Bacteroides.intestinalis
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:32.993024 INFO::Fitting model to feature number 14, Bacteroides.massiliensis
## 2024-05-01 19:45:33.159687 INFO::Fitting model to feature number 15, Bacteroides.ovatus
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:33.393388 INFO::Fitting model to feature number 16, Bacteroides.salyersiae
## 2024-05-01 19:45:33.618455 INFO::Fitting model to feature number 17, Bacteroides.stercoris
## 2024-05-01 19:45:33.829304 INFO::Fitting model to feature number 18, Bacteroides.thetaiotaomicron
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:33.998946 INFO::Fitting model to feature number 19, Bacteroides.uniformis
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:34.188773 INFO::Fitting model to feature number 20, Bacteroides.vulgatus
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:34.382579 INFO::Fitting model to feature number 21, Bacteroides.xylanisolvens
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:34.567341 INFO::Fitting model to feature number 22, Bacteroidales.bacterium.ph8
## 2024-05-01 19:45:34.732404 INFO::Fitting model to feature number 23, Barnesiella.intestinihominis
## 2024-05-01 19:45:34.890949 INFO::Fitting model to feature number 24, Coprobacter.fastidiosus
## 2024-05-01 19:45:35.107537 INFO::Fitting model to feature number 25, Odoribacter.splanchnicus
## 2024-05-01 19:45:35.614734 INFO::Fitting model to feature number 26, Parabacteroides.distasonis
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:35.838085 INFO::Fitting model to feature number 27, Parabacteroides.goldsteinii
## 2024-05-01 19:45:36.056121 INFO::Fitting model to feature number 28, Parabacteroides.merdae
## 2024-05-01 19:45:36.286346 INFO::Fitting model to feature number 29, Parabacteroides.unclassified
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:36.456834 INFO::Fitting model to feature number 30, Paraprevotella.clara
## 2024-05-01 19:45:36.61756 INFO::Fitting model to feature number 31, Paraprevotella.unclassified
## 2024-05-01 19:45:36.796092 INFO::Fitting model to feature number 32, Prevotella.copri
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:37.006531 INFO::Fitting model to feature number 33, Alistipes.finegoldii
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:37.196387 INFO::Fitting model to feature number 34, Alistipes.onderdonkii
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:37.355113 INFO::Fitting model to feature number 35, Alistipes.putredinis
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:37.55074 INFO::Fitting model to feature number 36, Alistipes.shahii
## 2024-05-01 19:45:37.685623 INFO::Fitting model to feature number 37, Alistipes.unclassified
## 2024-05-01 19:45:37.816236 INFO::Fitting model to feature number 38, Streptococcus.salivarius
## 2024-05-01 19:45:38.022941 INFO::Fitting model to feature number 39, Clostridium.bolteae
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:38.216075 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 19:45:38.324122 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 19:45:38.494074 INFO::Fitting model to feature number 41, Clostridium.clostridioforme
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:38.669372 INFO::Fitting model to feature number 42, Clostridium.hathewayi
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:38.849927 INFO::Fitting model to feature number 43, Clostridium.leptum
## 2024-05-01 19:45:39.085301 INFO::Fitting model to feature number 44, Clostridium.nexile
## 2024-05-01 19:45:39.29605 INFO::Fitting model to feature number 45, Clostridium.symbiosum
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:39.511041 INFO::Fitting model to feature number 46, Flavonifractor.plautii
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:39.726126 INFO::Fitting model to feature number 47, Eubacterium.eligens
## 2024-05-01 19:45:39.943991 INFO::Fitting model to feature number 48, Eubacterium.hallii
## 2024-05-01 19:45:40.155511 INFO::Fitting model to feature number 49, Eubacterium.rectale
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:40.368934 INFO::Fitting model to feature number 50, Eubacterium.siraeum
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:40.547355 INFO::Fitting model to feature number 51, Eubacterium.sp.3.1.31
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:40.722962 INFO::Fitting model to feature number 52, Eubacterium.ventriosum
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:40.844899 INFO::Fitting model to feature number 53, Ruminococcus.gnavus
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:40.985789 INFO::Fitting model to feature number 54, Ruminococcus.obeum
## 2024-05-01 19:45:41.140302 INFO::Fitting model to feature number 55, Ruminococcus.torques
## 2024-05-01 19:45:41.362818 INFO::Fitting model to feature number 56, Coprococcus.comes
## 2024-05-01 19:45:41.575985 INFO::Fitting model to feature number 57, Dorea.longicatena
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:41.790546 INFO::Fitting model to feature number 58, Lachnospiraceae.bacterium.1.1.57FAA
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:41.954056 INFO::Fitting model to feature number 59, Lachnospiraceae.bacterium.3.1.46FAA
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:42.084254 INFO::Fitting model to feature number 60, Roseburia.hominis
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:42.224157 INFO::Fitting model to feature number 61, Roseburia.intestinalis
## 2024-05-01 19:45:42.349109 INFO::Fitting model to feature number 62, Roseburia.inulinivorans
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:42.476431 INFO::Fitting model to feature number 63, Roseburia.unclassified
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:42.644489 INFO::Fitting model to feature number 64, Oscillibacter.unclassified
## 2024-05-01 19:45:42.779507 INFO::Fitting model to feature number 65, Peptostreptococcaceae.noname.unclassified
## 2024-05-01 19:45:42.913972 INFO::Fitting model to feature number 66, Faecalibacterium.prausnitzii
## 2024-05-01 19:45:43.04265 INFO::Fitting model to feature number 67, Ruminococcus.bromii
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:43.174809 INFO::Fitting model to feature number 68, Ruminococcus.callidus
## 2024-05-01 19:45:43.304769 INFO::Fitting model to feature number 69, Ruminococcus.lactaris
## 2024-05-01 19:45:43.462754 INFO::Fitting model to feature number 70, Subdoligranulum.unclassified
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:43.62364 INFO::Fitting model to feature number 71, Coprobacillus.unclassified
## 2024-05-01 19:45:43.777007 INFO::Fitting model to feature number 72, Acidaminococcus.unclassified
## 2024-05-01 19:45:43.99097 INFO::Fitting model to feature number 73, Dialister.invisus
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:44.13686 INFO::Fitting model to feature number 74, Veillonella.atypica
## 2024-05-01 19:45:44.28241 INFO::Fitting model to feature number 75, Veillonella.dispar
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:44.422837 INFO::Fitting model to feature number 76, Veillonella.parvula
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:44.573078 INFO::Fitting model to feature number 77, Veillonella.unclassified
## 2024-05-01 19:45:44.697733 INFO::Fitting model to feature number 78, Burkholderiales.bacterium.1.1.47
## 2024-05-01 19:45:44.830788 INFO::Fitting model to feature number 79, Parasutterella.excrementihominis
## 2024-05-01 19:45:44.983887 INFO::Fitting model to feature number 80, Sutterella.wadsworthensis
## 2024-05-01 19:45:45.120725 INFO::Fitting model to feature number 81, Bilophila.unclassified
## 2024-05-01 19:45:45.256247 INFO::Fitting model to feature number 82, Escherichia.coli
## 2024-05-01 19:45:45.399918 INFO::Fitting model to feature number 83, Escherichia.unclassified
## 2024-05-01 19:45:45.533433 INFO::Fitting model to feature number 84, Klebsiella.pneumoniae
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:45.688497 INFO::Fitting model to feature number 85, Haemophilus.parainfluenzae
## boundary (singular) fit: see help('isSingular')
## 2024-05-01 19:45:45.851782 INFO::Fitting model to feature number 86, Akkermansia.muciniphila
## 2024-05-01 19:45:46.062141 INFO::Fitting model to feature number 87, C2likevirus.unclassified
## 2024-05-01 19:45:46.269892 INFO::Counting total values for each feature
## 2024-05-01 19:45:46.319478 INFO::Writing filtered data to file demo_output/features/filtered_data.tsv
## 2024-05-01 19:45:46.622824 INFO::Writing filtered, normalized data to file demo_output/features/filtered_data_norm.tsv
## 2024-05-01 19:45:47.014013 INFO::Writing filtered, normalized, transformed data to file demo_output/features/filtered_data_norm_transformed.tsv
## 2024-05-01 19:45:47.443579 INFO::Writing residuals to file demo_output/fits/residuals.rds
## 2024-05-01 19:45:47.491405 INFO::Writing fitted values to file demo_output/fits/fitted.rds
## 2024-05-01 19:45:47.518567 INFO::Writing extracted random effects to file demo_output/fits/ranef.rds
## 2024-05-01 19:45:47.525111 INFO::Writing all results to file (ordered by increasing q-values): demo_output/all_results.tsv
## 2024-05-01 19:45:47.543191 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 19:45:47.555622 INFO::Writing heatmap of significant results to file: demo_output/heatmap.pdf
## 2024-05-01 19:45:47.761645 INFO::Writing association plots (one for each significant association) to output folder: demo_output
## 2024-05-01 19:45:47.77245 INFO::Plotting associations from most to least significant, grouped by metadata
## 2024-05-01 19:45:47.775456 INFO::Plotting data for metadata number 1, dysbiosisCD
## 2024-05-01 19:45:47.779553 INFO::Creating boxplot for categorical data, dysbiosisCD vs Faecalibacterium.prausnitzii
## 2024-05-01 19:45:48.161267 INFO::Creating boxplot for categorical data, dysbiosisCD vs Subdoligranulum.unclassified
## 2024-05-01 19:45:48.474441 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.uniformis
## 2024-05-01 19:45:48.789289 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.rectale
## 2024-05-01 19:45:49.083217 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.vulgatus
## 2024-05-01 19:45:49.340067 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.ovatus
## 2024-05-01 19:45:49.604923 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.obeum
## 2024-05-01 19:45:49.879801 INFO::Creating boxplot for categorical data, dysbiosisCD vs Oscillibacter.unclassified
## 2024-05-01 19:45:50.216826 INFO::Creating boxplot for categorical data, dysbiosisCD vs Roseburia.inulinivorans
## 2024-05-01 19:45:50.63218 INFO::Creating boxplot for categorical data, dysbiosisCD vs Roseburia.hominis
## 2024-05-01 19:45:50.951023 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.thetaiotaomicron
## 2024-05-01 19:45:51.304974 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.putredinis
## 2024-05-01 19:45:51.672994 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.distasonis
## 2024-05-01 19:45:52.069928 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.dorei
## 2024-05-01 19:45:52.485367 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.shahii
## 2024-05-01 19:45:52.775938 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.xylanisolvens
## 2024-05-01 19:45:53.55184 INFO::Creating boxplot for categorical data, dysbiosisCD vs Clostridium.leptum
## 2024-05-01 19:45:53.86037 INFO::Creating boxplot for categorical data, dysbiosisCD vs Dorea.longicatena
## 2024-05-01 19:45:54.272496 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.caccae
## 2024-05-01 19:45:54.680996 INFO::Creating boxplot for categorical data, dysbiosisCD vs Lachnospiraceae.bacterium.3.1.46FAA
## 2024-05-01 19:45:55.09779 INFO::Creating boxplot for categorical data, dysbiosisCD vs Escherichia.coli
## 2024-05-01 19:45:55.424029 INFO::Creating boxplot for categorical data, dysbiosisCD vs Klebsiella.pneumoniae
## 2024-05-01 19:45:55.98745 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bilophila.unclassified
## 2024-05-01 19:45:56.331871 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.finegoldii
## 2024-05-01 19:45:56.603995 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.eligens
## 2024-05-01 19:45:56.87459 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.stercoris
## 2024-05-01 19:45:57.228012 INFO::Creating boxplot for categorical data, dysbiosisCD vs Coprococcus.comes
## 2024-05-01 19:45:57.481492 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.fragilis
## 2024-05-01 19:45:57.788434 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.finegoldii
## 2024-05-01 19:45:58.055339 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.hallii
## 2024-05-01 19:45:58.417416 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.faecis
## 2024-05-01 19:45:59.167319 INFO::Creating boxplot for categorical data, dysbiosisCD vs Paraprevotella.clara
## 2024-05-01 19:45:59.555998 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.siraeum
## 2024-05-01 19:45:59.912059 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.merdae
## 2024-05-01 19:46:00.241773 INFO::Creating boxplot for categorical data, dysbiosisCD vs Paraprevotella.unclassified
## 2024-05-01 19:46:00.556046 INFO::Creating boxplot for categorical data, dysbiosisCD vs Collinsella.aerofaciens
## 2024-05-01 19:46:00.904741 INFO::Creating boxplot for categorical data, dysbiosisCD vs Odoribacter.splanchnicus
## 2024-05-01 19:46:01.245943 INFO::Creating boxplot for categorical data, dysbiosisCD vs Clostridium.clostridioforme
## 2024-05-01 19:46:01.59251 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.eggerthii
## 2024-05-01 19:46:01.923 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.onderdonkii
## 2024-05-01 19:46:02.168292 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.ventriosum
## 2024-05-01 19:46:02.430572 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.lactaris
## 2024-05-01 19:46:02.686998 INFO::Creating boxplot for categorical data, dysbiosisCD vs Burkholderiales.bacterium.1.1.47
## 2024-05-01 19:46:02.939059 INFO::Creating boxplot for categorical data, dysbiosisCD vs Dialister.invisus
## 2024-05-01 19:46:03.187987 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.bromii
## 2024-05-01 19:46:03.455997 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parasutterella.excrementihominis
## 2024-05-01 19:46:03.773235 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.unclassified
## 2024-05-01 19:46:04.049679 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.torques
## 2024-05-01 19:46:04.290791 INFO::Creating boxplot for categorical data, dysbiosisCD vs Coprobacillus.unclassified
## 2024-05-01 19:46:04.592791 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.cellulosilyticus
## 2024-05-01 19:46:04.875862 INFO::Creating boxplot for categorical data, dysbiosisCD vs Roseburia.intestinalis
## 2024-05-01 19:46:05.196503 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.unclassified
## 2024-05-01 19:46:05.552037 INFO::Creating boxplot for categorical data, dysbiosisCD vs Acidaminococcus.unclassified
## 2024-05-01 19:46:05.9109 INFO::Creating boxplot for categorical data, dysbiosisCD vs Barnesiella.intestinihominis
## 2024-05-01 19:46:06.240078 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.massiliensis
## 2024-05-01 19:46:06.582607 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.sp.3.1.31
## 2024-05-01 19:46:06.839213 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.salyersiae
## 2024-05-01 19:46:07.090858 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroidales.bacterium.ph8
## 2024-05-01 19:46:07.333545 INFO::Creating boxplot for categorical data, dysbiosisCD vs Clostridium.citroniae
## 2024-05-01 19:46:07.610829 INFO::Creating boxplot for categorical data, dysbiosisCD vs Flavonifractor.plautii
## 2024-05-01 19:46:08.006083 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.goldsteinii
## 2024-05-01 19:46:08.417592 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bifidobacterium.longum
## 2024-05-01 19:46:08.789312 INFO::Creating boxplot for categorical data, dysbiosisCD vs Lachnospiraceae.bacterium.1.1.57FAA
## 2024-05-01 19:46:13.132584 INFO::Plotting data for metadata number 2, dysbiosisUC
## 2024-05-01 19:46:13.137109 INFO::Creating boxplot for categorical data, dysbiosisUC vs Subdoligranulum.unclassified
## 2024-05-01 19:46:13.45538 INFO::Creating boxplot for categorical data, dysbiosisUC vs Faecalibacterium.prausnitzii
## 2024-05-01 19:46:13.749878 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.caccae
## 2024-05-01 19:46:14.041508 INFO::Creating boxplot for categorical data, dysbiosisUC vs Oscillibacter.unclassified
## 2024-05-01 19:46:14.306409 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.uniformis
## 2024-05-01 19:46:14.733346 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.siraeum
## 2024-05-01 19:46:15.163155 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.ovatus
## 2024-05-01 19:46:15.486067 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.fragilis
## 2024-05-01 19:46:15.888699 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.shahii
## 2024-05-01 19:46:16.22352 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.rectale
## 2024-05-01 19:46:16.472137 INFO::Creating boxplot for categorical data, dysbiosisUC vs Roseburia.hominis
## 2024-05-01 19:46:16.767002 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.putredinis
## 2024-05-01 19:46:17.116918 INFO::Creating boxplot for categorical data, dysbiosisUC vs Lachnospiraceae.bacterium.3.1.46FAA
## 2024-05-01 19:46:17.497828 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.hallii
## 2024-05-01 19:46:17.845614 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.xylanisolvens
## 2024-05-01 19:46:18.106034 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.stercoris
## 2024-05-01 19:46:18.38182 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.finegoldii
## 2024-05-01 19:46:18.635967 INFO::Creating boxplot for categorical data, dysbiosisUC vs Barnesiella.intestinihominis
## 2024-05-01 19:46:19.018506 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.leptum
## 2024-05-01 19:46:19.369165 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.gnavus
## 2024-05-01 19:46:19.66352 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.onderdonkii
## 2024-05-01 19:46:19.938249 INFO::Creating boxplot for categorical data, dysbiosisUC vs Flavonifractor.plautii
## 2024-05-01 19:46:20.463853 INFO::Creating boxplot for categorical data, dysbiosisUC vs Parabacteroides.merdae
## 2024-05-01 19:46:20.800536 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.cellulosilyticus
## 2024-05-01 19:46:21.184142 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bifidobacterium.longum
## 2024-05-01 19:46:21.478082 INFO::Creating boxplot for categorical data, dysbiosisUC vs Klebsiella.pneumoniae
## 2024-05-01 19:46:21.747581 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.citroniae
## 2024-05-01 19:46:21.990258 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.ventriosum
## 2024-05-01 19:46:22.277627 INFO::Creating boxplot for categorical data, dysbiosisUC vs Parabacteroides.distasonis
## 2024-05-01 19:46:22.546753 INFO::Creating boxplot for categorical data, dysbiosisUC vs Parabacteroides.goldsteinii
## 2024-05-01 19:46:22.871155 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.torques
## 2024-05-01 19:46:23.258653 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroidales.bacterium.ph8
## 2024-05-01 19:46:23.579752 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.obeum
## 2024-05-01 19:46:23.824811 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bifidobacterium.bifidum
## 2024-05-01 19:46:24.142816 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bifidobacterium.adolescentis
## 2024-05-01 19:46:24.385292 INFO::Creating boxplot for categorical data, dysbiosisUC vs Collinsella.aerofaciens
## 2024-05-01 19:46:24.655106 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.hathewayi
## 2024-05-01 19:46:24.915929 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bilophila.unclassified
## 2024-05-01 19:46:25.168081 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.eligens
## 2024-05-01 19:46:25.427731 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.vulgatus
## 2024-05-01 19:46:25.67725 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.bolteae
## 2024-05-01 19:46:26.024162 INFO::Creating boxplot for categorical data, dysbiosisUC vs Dialister.invisus
## 2024-05-01 19:46:26.303061 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.lactaris
## 2024-05-01 19:46:26.672082 INFO::Creating boxplot for categorical data, dysbiosisUC vs Burkholderiales.bacterium.1.1.47
## 2024-05-01 19:46:30.639596 INFO::Plotting data for metadata number 3, dysbiosisnonIBD
## 2024-05-01 19:46:30.643408 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Faecalibacterium.prausnitzii
## 2024-05-01 19:46:30.963443 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.rectale
## 2024-05-01 19:46:31.356891 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.sp.3.1.31
## 2024-05-01 19:46:31.730681 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.hominis
## 2024-05-01 19:46:31.98943 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Subdoligranulum.unclassified
## 2024-05-01 19:46:32.291312 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Lachnospiraceae.bacterium.3.1.46FAA
## 2024-05-01 19:46:32.551691 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Ruminococcus.torques
## 2024-05-01 19:46:32.830089 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Clostridium.leptum
## 2024-05-01 19:46:33.090516 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Odoribacter.splanchnicus
## 2024-05-01 19:46:33.371654 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Escherichia.coli
## 2024-05-01 19:46:33.65947 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Ruminococcus.obeum
## 2024-05-01 19:46:34.036757 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bilophila.unclassified
## 2024-05-01 19:46:34.407743 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.uniformis
## 2024-05-01 19:46:34.761251 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.eligens
## 2024-05-01 19:46:35.120907 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Klebsiella.pneumoniae
## 2024-05-01 19:46:35.409094 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Ruminococcus.bromii
## 2024-05-01 19:46:35.656053 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.vulgatus
## 2024-05-01 19:46:35.920473 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs C2likevirus.unclassified
## 2024-05-01 19:46:36.178719 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bifidobacterium.adolescentis
## 2024-05-01 19:46:36.446136 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Alistipes.finegoldii
## 2024-05-01 19:46:36.708347 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.inulinivorans
## 2024-05-01 19:46:36.983576 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Alistipes.onderdonkii
## 2024-05-01 19:46:37.245524 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.unclassified
## 2024-05-01 19:46:37.518561 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.hallii
## 2024-05-01 19:46:37.786716 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.intestinalis
## 2024-05-01 19:46:38.076112 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Prevotella.copri
## 2024-05-01 19:46:38.401299 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.dorei
## 2024-05-01 19:46:38.703602 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.fragilis
## 2024-05-01 19:46:38.988919 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Alistipes.shahii
## 2024-05-01 19:46:43.985096 INFO::Plotting data for metadata number 4, antibiotics
## 2024-05-01 19:46:43.988264 INFO::Creating boxplot for categorical data, antibiotics vs Roseburia.inulinivorans
## 2024-05-01 19:46:44.258547 INFO::Creating boxplot for categorical data, antibiotics vs Roseburia.hominis
## 2024-05-01 19:46:44.53776 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.rectale
## 2024-05-01 19:46:44.891449 INFO::Creating boxplot for categorical data, antibiotics vs Dialister.invisus
## 2024-05-01 19:46:45.168901 INFO::Creating boxplot for categorical data, antibiotics vs Roseburia.intestinalis
## 2024-05-01 19:46:45.496944 INFO::Creating boxplot for categorical data, antibiotics vs Dorea.longicatena
## 2024-05-01 19:46:45.75695 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.callidus
## 2024-05-01 19:46:46.050019 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.bromii
## 2024-05-01 19:46:46.305101 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.obeum
## 2024-05-01 19:46:46.552286 INFO::Creating boxplot for categorical data, antibiotics vs Klebsiella.pneumoniae
## 2024-05-01 19:46:46.824632 INFO::Creating boxplot for categorical data, antibiotics vs Bifidobacterium.adolescentis
## 2024-05-01 19:46:47.057011 INFO::Creating boxplot for categorical data, antibiotics vs Faecalibacterium.prausnitzii
## 2024-05-01 19:46:47.312233 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.hallii
## 2024-05-01 19:46:47.55891 INFO::Creating boxplot for categorical data, antibiotics vs Bilophila.unclassified
## 2024-05-01 19:46:47.836489 INFO::Creating boxplot for categorical data, antibiotics vs Clostridium.leptum
## 2024-05-01 19:46:48.078542 INFO::Creating boxplot for categorical data, antibiotics vs Lachnospiraceae.bacterium.3.1.46FAA
## 2024-05-01 19:46:48.337205 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.finegoldii
## 2024-05-01 19:46:48.589836 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.sp.3.1.31
## 2024-05-01 19:46:48.854501 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.fragilis
## 2024-05-01 19:46:49.092844 INFO::Creating boxplot for categorical data, antibiotics vs Alistipes.onderdonkii
## 2024-05-01 19:46:49.403709 INFO::Creating boxplot for categorical data, antibiotics vs Sutterella.wadsworthensis
## 2024-05-01 19:46:49.73166 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.eligens
## 2024-05-01 19:46:50.143309 INFO::Creating boxplot for categorical data, antibiotics vs Collinsella.aerofaciens
## 2024-05-01 19:46:50.455921 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.thetaiotaomicron
## 2024-05-01 19:46:50.735168 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.eggerthii
## 2024-05-01 19:46:51.007196 INFO::Creating boxplot for categorical data, antibiotics vs Haemophilus.parainfluenzae
## 2024-05-01 19:46:51.263466 INFO::Creating boxplot for categorical data, antibiotics vs Bifidobacterium.pseudocatenulatum
## 2024-05-01 19:46:51.557624 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.torques
## 2024-05-01 19:46:51.82461 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.ventriosum
## 2024-05-01 19:46:52.126697 INFO::Creating boxplot for categorical data, antibiotics vs Parasutterella.excrementihominis
## 2024-05-01 19:46:52.399017 INFO::Creating boxplot for categorical data, antibiotics vs Peptostreptococcaceae.noname.unclassified
## 2024-05-01 19:46:52.723 INFO::Creating boxplot for categorical data, antibiotics vs Veillonella.dispar
## 2024-05-01 19:46:53.041104 INFO::Creating boxplot for categorical data, antibiotics vs Veillonella.atypica
## 2024-05-01 19:46:53.317088 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroidales.bacterium.ph8
## 2024-05-01 19:46:53.971735 INFO::Creating boxplot for categorical data, antibiotics vs Clostridium.nexile
## 2024-05-01 19:46:54.229672 INFO::Creating boxplot for categorical data, antibiotics vs Burkholderiales.bacterium.1.1.47
## 2024-05-01 19:46:54.466344 INFO::Creating boxplot for categorical data, antibiotics vs Lachnospiraceae.bacterium.1.1.57FAA
## 2024-05-01 19:46:54.732449 INFO::Creating boxplot for categorical data, antibiotics vs Akkermansia.muciniphila
## 2024-05-01 19:46:54.961736 INFO::Creating boxplot for categorical data, antibiotics vs Clostridium.citroniae
## 2024-05-01 19:46:55.267522 INFO::Creating boxplot for categorical data, antibiotics vs Odoribacter.splanchnicus
## 2024-05-01 19:47:01.941184 INFO::Plotting data for metadata number 5, age
## 2024-05-01 19:47:01.945726 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 19:47:02.375098 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 19:47:02.786098 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 19:47:03.031868 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 19:47:03.329999 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 19:47:03.606287 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 19:47:03.888418 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 19:47:04.178762 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 19:47:04.439097 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 19:47:04.731995 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 19:47:04.992654 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 19:47:05.27743 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 19:47:05.536081 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 19:47:05.854695 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 19:47:06.150377 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 19:47:06.397353 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 19:47:06.702643 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 19:47:06.948044 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 19:47:07.239265 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 19:47:07.563364 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 19:47:07.848793 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 19:47:08.133632 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 19:47:12.328328 INFO::Plotting data for metadata number 6, diagnosis
## 2024-05-01 19:47:12.331695 INFO::Creating boxplot for categorical data, diagnosis vs Bifidobacterium.adolescentis
## 2024-05-01 19:47:12.626734 INFO::Creating boxplot for categorical data, diagnosis vs Akkermansia.muciniphila
## 2024-05-01 19:47:12.872305 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.bolteae
## 2024-05-01 19:47:13.194317 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.putredinis
## 2024-05-01 19:47:13.472584 INFO::Creating boxplot for categorical data, diagnosis vs Coprobacillus.unclassified
## 2024-05-01 19:47:13.779362 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.clostridioforme
## 2024-05-01 19:47:14.024654 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.shahii
## 2024-05-01 19:47:14.279338 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.bromii
## 2024-05-01 19:47:14.575682 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.leptum
## 2024-05-01 19:47:14.834342 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.symbiosum
## 2024-05-01 19:47:15.073498 INFO::Creating boxplot for categorical data, diagnosis vs Roseburia.inulinivorans
## 2024-05-01 19:47:15.320717 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.bromii
## 2024-05-01 19:47:15.560507 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.shahii
## 2024-05-01 19:47:15.817462 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.finegoldii
## 2024-05-01 19:47:16.070752 INFO::Creating boxplot for categorical data, diagnosis vs Parabacteroides.goldsteinii
## 2024-05-01 19:47:16.316874 INFO::Creating boxplot for categorical data, diagnosis vs Bilophila.unclassified
## 2024-05-01 19:47:16.561295 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.leptum
## 2024-05-01 19:47:16.810481 INFO::Creating boxplot for categorical data, diagnosis vs Roseburia.hominis
## 2024-05-01 19:47:17.048145 INFO::Creating boxplot for categorical data, diagnosis vs Coprobacillus.unclassified
## 2024-05-01 19:47:17.330062 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.onderdonkii
## 2024-05-01 19:47:17.630403 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.ventriosum
## 2024-05-01 19:47:17.977807 INFO::Creating boxplot for categorical data, diagnosis vs Roseburia.hominis
## 2024-05-01 19:47:18.233928 INFO::Creating boxplot for categorical data, diagnosis vs Sutterella.wadsworthensis
## 2024-05-01 19:47:18.504324 INFO::Creating boxplot for categorical data, diagnosis vs Akkermansia.muciniphila
## 2024-05-01 19:47:18.853113 INFO::Creating boxplot for categorical data, diagnosis vs Sutterella.wadsworthensis
## 2024-05-01 19:47:19.240778 INFO::Creating boxplot for categorical data, diagnosis vs Subdoligranulum.unclassified
## 2024-05-01 19:47:19.603619 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.symbiosum
## 2024-05-01 19:47:19.959726 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.onderdonkii
## 2024-05-01 19:47:20.289101 INFO::Creating boxplot for categorical data, diagnosis vs Parabacteroides.unclassified
## 2024-05-01 19:47:20.614072 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.lactaris
## 2024-05-01 19:47:20.915081 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.callidus
## 2024-05-01 19:47:21.181597 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.gnavus
## 2024-05-01 19:47:21.444792 INFO::Creating boxplot for categorical data, diagnosis vs Bacteroides.fragilis
## 2024-05-01 19:47:21.809189 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.rectale
## 2024-05-01 19:47:22.082555 INFO::Creating boxplot for categorical data, diagnosis vs Odoribacter.splanchnicus
## 2024-05-01 19:47:22.342395 INFO::Creating boxplot for categorical data, diagnosis vs Parabacteroides.distasonis
## 2024-05-01 19:47:22.618558 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.rectale
## 2024-05-01 19:47:22.866522 INFO::Creating boxplot for categorical data, diagnosis vs Bacteroides.finegoldii
## 2024-05-01 19:47:23.172554 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.callidus
## 2024-05-01 19:47:23.419667 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.finegoldii
## 2024-05-01 19:47:23.735732 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.putredinis
## 2024-05-01 19:47:24.123174 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.bolteae
## 2024-05-01 19:47:24.406375 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.siraeum
Session Info

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

sessionInfo()
## R version 4.4.0 beta (2024-04-15 r86425 ucrt)
## Platform: x86_64-w64-mingw32/x64
## Running under: Windows Server 2022 x64 (build 20348)
## 
## Matrix products: default
## 
## 
## locale:
## [1] LC_COLLATE=C                          
## [2] LC_CTYPE=English_United States.utf8   
## [3] LC_MONETARY=English_United States.utf8
## [4] LC_NUMERIC=C                          
## [5] LC_TIME=English_United States.utf8    
## 
## time zone: America/New_York
## tzcode source: internal
## 
## attached base packages:
## [1] stats     graphics  grDevices utils     datasets  methods   base     
## 
## other attached packages:
## [1] Maaslin2_1.18.0
## 
## 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.