MaAsLin2 User Manual

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

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

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

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

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

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


Description

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

Requirements

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

Installation

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

From command line

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

From R

Install Bioconductor and then install Maaslin2

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

How to Run

MaAsLin2 can be run from the command line or as an R function. Both methods require the same arguments, have the same options, and use the same default settings.

Input Files

MaAsLin2 requires two input files.

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

The data file can contain samples not included in the metadata file (along with the reverse case). For both cases, those samples not included in both files will be removed from the analysis. Also the samples do not need to be in the same order in the two files.

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

Output Files

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

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

Run a Demo

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

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

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

Command line

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

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

In R

library(Maaslin2)
input_data <- system.file(
    'extdata','HMP2_taxonomy.tsv', package="Maaslin2")
input_metadata <-system.file(
    'extdata','HMP2_metadata.tsv', package="Maaslin2")
fit_data <- Maaslin2(
    input_data, input_metadata, 'demo_output',
    fixed_effects = c('diagnosis', 'dysbiosisnonIBD','dysbiosisUC','dysbiosisCD', 'antibiotics', 'age'),
    random_effects = c('site', 'subject'),
    reference = "diagnosis,nonIBD",
    standardize = FALSE)
## [1] "Creating output folder"
## [1] "Creating output feature tables folder"
## [1] "Creating output fits folder"
## [1] "Creating output figures folder"
## 2023-10-24 21:01:18.601238 INFO::Writing function arguments to log file
## 2023-10-24 21:01:18.648773 INFO::Verifying options selected are valid
## 2023-10-24 21:01:18.703382 INFO::Determining format of input files
## 2023-10-24 21:01:18.706242 INFO::Input format is data samples as rows and metadata samples as rows
## 2023-10-24 21:01:18.716258 INFO::Formula for random effects: expr ~ (1 | site) + (1 | subject)
## 2023-10-24 21:01:18.718781 INFO::Formula for fixed effects: expr ~  diagnosis + dysbiosisnonIBD + dysbiosisUC + dysbiosisCD + antibiotics + age
## 2023-10-24 21:01:18.722674 INFO::Filter data based on min abundance and min prevalence
## 2023-10-24 21:01:18.724731 INFO::Total samples in data: 1595
## 2023-10-24 21:01:18.726755 INFO::Min samples required with min abundance for a feature not to be filtered: 159.500000
## 2023-10-24 21:01:18.73374 INFO::Total filtered features: 0
## 2023-10-24 21:01:18.735988 INFO::Filtered feature names from abundance and prevalence filtering:
## 2023-10-24 21:01:18.747226 INFO::Total filtered features with variance filtering: 0
## 2023-10-24 21:01:18.749511 INFO::Filtered feature names from variance filtering:
## 2023-10-24 21:01:18.751492 INFO::Running selected normalization method: TSS
## 2023-10-24 21:01:19.839007 INFO::Bypass z-score application to metadata
## 2023-10-24 21:01:19.840895 INFO::Running selected transform method: LOG
## 2023-10-24 21:01:19.861036 INFO::Running selected analysis method: LM
## 2023-10-24 21:01:20.445206 INFO::Fitting model to feature number 1, Bifidobacterium.adolescentis
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:20.790822 INFO::Fitting model to feature number 2, Bifidobacterium.bifidum
## 2023-10-24 21:01:20.968382 INFO::Fitting model to feature number 3, Bifidobacterium.longum
## 2023-10-24 21:01:21.126303 INFO::Fitting model to feature number 4, Bifidobacterium.pseudocatenulatum
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:21.254053 INFO::Fitting model to feature number 5, Collinsella.aerofaciens
## 2023-10-24 21:01:21.402257 INFO::Fitting model to feature number 6, Bacteroides.caccae
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:21.548459 INFO::Fitting model to feature number 7, Bacteroides.cellulosilyticus
## 2023-10-24 21:01:21.714591 INFO::Fitting model to feature number 8, Bacteroides.dorei
## 2023-10-24 21:01:21.858136 INFO::Fitting model to feature number 9, Bacteroides.eggerthii
## 2023-10-24 21:01:21.992902 INFO::Fitting model to feature number 10, Bacteroides.faecis
## 2023-10-24 21:01:22.154827 INFO::Fitting model to feature number 11, Bacteroides.finegoldii
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:22.285349 INFO::Fitting model to feature number 12, Bacteroides.fragilis
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:22.419696 INFO::Fitting model to feature number 13, Bacteroides.intestinalis
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:22.559399 INFO::Fitting model to feature number 14, Bacteroides.massiliensis
## 2023-10-24 21:01:22.701557 INFO::Fitting model to feature number 15, Bacteroides.ovatus
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:22.832668 INFO::Fitting model to feature number 16, Bacteroides.salyersiae
## 2023-10-24 21:01:22.96979 INFO::Fitting model to feature number 17, Bacteroides.stercoris
## 2023-10-24 21:01:23.117822 INFO::Fitting model to feature number 18, Bacteroides.thetaiotaomicron
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:23.255476 INFO::Fitting model to feature number 19, Bacteroides.uniformis
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:23.383989 INFO::Fitting model to feature number 20, Bacteroides.vulgatus
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:23.523291 INFO::Fitting model to feature number 21, Bacteroides.xylanisolvens
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:23.67596 INFO::Fitting model to feature number 22, Bacteroidales.bacterium.ph8
## 2023-10-24 21:01:23.843157 INFO::Fitting model to feature number 23, Barnesiella.intestinihominis
## 2023-10-24 21:01:23.996301 INFO::Fitting model to feature number 24, Coprobacter.fastidiosus
## 2023-10-24 21:01:24.136646 INFO::Fitting model to feature number 25, Odoribacter.splanchnicus
## 2023-10-24 21:01:24.280558 INFO::Fitting model to feature number 26, Parabacteroides.distasonis
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:24.412961 INFO::Fitting model to feature number 27, Parabacteroides.goldsteinii
## 2023-10-24 21:01:24.566177 INFO::Fitting model to feature number 28, Parabacteroides.merdae
## 2023-10-24 21:01:24.74071 INFO::Fitting model to feature number 29, Parabacteroides.unclassified
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:24.903545 INFO::Fitting model to feature number 30, Paraprevotella.clara
## 2023-10-24 21:01:25.043026 INFO::Fitting model to feature number 31, Paraprevotella.unclassified
## 2023-10-24 21:01:25.173291 INFO::Fitting model to feature number 32, Prevotella.copri
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:25.31398 INFO::Fitting model to feature number 33, Alistipes.finegoldii
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:25.447136 INFO::Fitting model to feature number 34, Alistipes.onderdonkii
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:25.580429 INFO::Fitting model to feature number 35, Alistipes.putredinis
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:25.714176 INFO::Fitting model to feature number 36, Alistipes.shahii
## 2023-10-24 21:01:25.854302 INFO::Fitting model to feature number 37, Alistipes.unclassified
## 2023-10-24 21:01:26.007914 INFO::Fitting model to feature number 38, Streptococcus.salivarius
## 2023-10-24 21:01:26.148914 INFO::Fitting model to feature number 39, Clostridium.bolteae
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:26.294838 INFO::Fitting model to feature number 40, Clostridium.citroniae
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:26.425056 INFO::Fitting model to feature number 41, Clostridium.clostridioforme
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:26.576057 INFO::Fitting model to feature number 42, Clostridium.hathewayi
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:26.746524 INFO::Fitting model to feature number 43, Clostridium.leptum
## 2023-10-24 21:01:26.898373 INFO::Fitting model to feature number 44, Clostridium.nexile
## 2023-10-24 21:01:27.047054 INFO::Fitting model to feature number 45, Clostridium.symbiosum
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:27.175305 INFO::Fitting model to feature number 46, Flavonifractor.plautii
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:27.334554 INFO::Fitting model to feature number 47, Eubacterium.eligens
## 2023-10-24 21:01:27.48946 INFO::Fitting model to feature number 48, Eubacterium.hallii
## 2023-10-24 21:01:27.638988 INFO::Fitting model to feature number 49, Eubacterium.rectale
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:27.786598 INFO::Fitting model to feature number 50, Eubacterium.siraeum
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:27.919949 INFO::Fitting model to feature number 51, Eubacterium.sp.3.1.31
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:28.056462 INFO::Fitting model to feature number 52, Eubacterium.ventriosum
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:28.179324 INFO::Fitting model to feature number 53, Ruminococcus.gnavus
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:28.330048 INFO::Fitting model to feature number 54, Ruminococcus.obeum
## 2023-10-24 21:01:28.459571 INFO::Fitting model to feature number 55, Ruminococcus.torques
## 2023-10-24 21:01:28.601397 INFO::Fitting model to feature number 56, Coprococcus.comes
## 2023-10-24 21:01:28.760553 INFO::Fitting model to feature number 57, Dorea.longicatena
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:28.901535 INFO::Fitting model to feature number 58, Lachnospiraceae.bacterium.1.1.57FAA
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:29.050796 INFO::Fitting model to feature number 59, Lachnospiraceae.bacterium.3.1.46FAA
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:29.203695 INFO::Fitting model to feature number 60, Roseburia.hominis
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:29.353711 INFO::Fitting model to feature number 61, Roseburia.intestinalis
## 2023-10-24 21:01:29.525574 INFO::Fitting model to feature number 62, Roseburia.inulinivorans
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:29.668003 INFO::Fitting model to feature number 63, Roseburia.unclassified
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:29.802235 INFO::Fitting model to feature number 64, Oscillibacter.unclassified
## 2023-10-24 21:01:29.96353 INFO::Fitting model to feature number 65, Peptostreptococcaceae.noname.unclassified
## 2023-10-24 21:01:30.093904 INFO::Fitting model to feature number 66, Faecalibacterium.prausnitzii
## 2023-10-24 21:01:30.22821 INFO::Fitting model to feature number 67, Ruminococcus.bromii
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:30.362372 INFO::Fitting model to feature number 68, Ruminococcus.callidus
## 2023-10-24 21:01:30.488364 INFO::Fitting model to feature number 69, Ruminococcus.lactaris
## 2023-10-24 21:01:30.655371 INFO::Fitting model to feature number 70, Subdoligranulum.unclassified
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:30.802099 INFO::Fitting model to feature number 71, Coprobacillus.unclassified
## 2023-10-24 21:01:30.92652 INFO::Fitting model to feature number 72, Acidaminococcus.unclassified
## 2023-10-24 21:01:31.073048 INFO::Fitting model to feature number 73, Dialister.invisus
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:31.208803 INFO::Fitting model to feature number 74, Veillonella.atypica
## 2023-10-24 21:01:31.355422 INFO::Fitting model to feature number 75, Veillonella.dispar
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:31.493981 INFO::Fitting model to feature number 76, Veillonella.parvula
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:31.648231 INFO::Fitting model to feature number 77, Veillonella.unclassified
## 2023-10-24 21:01:31.815983 INFO::Fitting model to feature number 78, Burkholderiales.bacterium.1.1.47
## 2023-10-24 21:01:31.983748 INFO::Fitting model to feature number 79, Parasutterella.excrementihominis
## 2023-10-24 21:01:32.181398 INFO::Fitting model to feature number 80, Sutterella.wadsworthensis
## 2023-10-24 21:01:32.377949 INFO::Fitting model to feature number 81, Bilophila.unclassified
## 2023-10-24 21:01:32.5414 INFO::Fitting model to feature number 82, Escherichia.coli
## 2023-10-24 21:01:32.708574 INFO::Fitting model to feature number 83, Escherichia.unclassified
## 2023-10-24 21:01:32.834812 INFO::Fitting model to feature number 84, Klebsiella.pneumoniae
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:32.97062 INFO::Fitting model to feature number 85, Haemophilus.parainfluenzae
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 21:01:33.10005 INFO::Fitting model to feature number 86, Akkermansia.muciniphila
## 2023-10-24 21:01:33.244528 INFO::Fitting model to feature number 87, C2likevirus.unclassified
## 2023-10-24 21:01:33.513295 INFO::Counting total values for each feature
## 2023-10-24 21:01:33.548323 INFO::Writing filtered data to file demo_output/features/filtered_data.tsv
## 2023-10-24 21:01:33.846532 INFO::Writing filtered, normalized data to file demo_output/features/filtered_data_norm.tsv
## 2023-10-24 21:01:34.166802 INFO::Writing filtered, normalized, transformed data to file demo_output/features/filtered_data_norm_transformed.tsv
## 2023-10-24 21:01:34.54452 INFO::Writing residuals to file demo_output/fits/residuals.rds
## 2023-10-24 21:01:34.591541 INFO::Writing fitted values to file demo_output/fits/fitted.rds
## 2023-10-24 21:01:34.617802 INFO::Writing extracted random effects to file demo_output/fits/ranef.rds
## 2023-10-24 21:01:34.624472 INFO::Writing all results to file (ordered by increasing q-values): demo_output/all_results.tsv
## 2023-10-24 21:01:34.639114 INFO::Writing the significant results (those which are less than or equal to the threshold of 0.250000 ) to file (ordered by increasing q-values): demo_output/significant_results.tsv
## 2023-10-24 21:01:34.646253 INFO::Writing heatmap of significant results to file: demo_output/heatmap.pdf
## 2023-10-24 21:01:34.864623 INFO::Writing association plots (one for each significant association) to output folder: demo_output
## 2023-10-24 21:01:34.87776 INFO::Plotting associations from most to least significant, grouped by metadata
## 2023-10-24 21:01:34.880894 INFO::Plotting data for metadata number 1, dysbiosisCD
## 2023-10-24 21:01:34.885184 INFO::Creating boxplot for categorical data, dysbiosisCD vs Faecalibacterium.prausnitzii
## 2023-10-24 21:01:35.253735 INFO::Creating boxplot for categorical data, dysbiosisCD vs Subdoligranulum.unclassified
## 2023-10-24 21:01:35.539182 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.uniformis
## 2023-10-24 21:01:35.810606 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.rectale
## 2023-10-24 21:01:36.091301 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.vulgatus
## 2023-10-24 21:01:36.354271 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.ovatus
## 2023-10-24 21:01:36.663232 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.obeum
## 2023-10-24 21:01:36.930385 INFO::Creating boxplot for categorical data, dysbiosisCD vs Oscillibacter.unclassified
## 2023-10-24 21:01:37.469427 INFO::Creating boxplot for categorical data, dysbiosisCD vs Roseburia.inulinivorans
## 2023-10-24 21:01:37.736007 INFO::Creating boxplot for categorical data, dysbiosisCD vs Roseburia.hominis
## 2023-10-24 21:01:38.00181 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.thetaiotaomicron
## 2023-10-24 21:01:38.26275 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.putredinis
## 2023-10-24 21:01:38.537077 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.distasonis
## 2023-10-24 21:01:38.84804 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.dorei
## 2023-10-24 21:01:39.105387 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.shahii
## 2023-10-24 21:01:39.37717 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.xylanisolvens
## 2023-10-24 21:01:39.672517 INFO::Creating boxplot for categorical data, dysbiosisCD vs Clostridium.leptum
## 2023-10-24 21:01:40.008477 INFO::Creating boxplot for categorical data, dysbiosisCD vs Dorea.longicatena
## 2023-10-24 21:01:40.302399 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.caccae
## 2023-10-24 21:01:40.547295 INFO::Creating boxplot for categorical data, dysbiosisCD vs Lachnospiraceae.bacterium.3.1.46FAA
## 2023-10-24 21:01:40.828862 INFO::Creating boxplot for categorical data, dysbiosisCD vs Escherichia.coli
## 2023-10-24 21:01:41.075014 INFO::Creating boxplot for categorical data, dysbiosisCD vs Klebsiella.pneumoniae
## 2023-10-24 21:01:41.361876 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bilophila.unclassified
## 2023-10-24 21:01:41.615641 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.finegoldii
## 2023-10-24 21:01:41.882051 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.eligens
## 2023-10-24 21:01:42.131867 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.stercoris
## 2023-10-24 21:01:42.397034 INFO::Creating boxplot for categorical data, dysbiosisCD vs Coprococcus.comes
## 2023-10-24 21:01:42.687245 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.fragilis
## 2023-10-24 21:01:42.93843 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.finegoldii
## 2023-10-24 21:01:43.202388 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.hallii
## 2023-10-24 21:01:43.448103 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.faecis
## 2023-10-24 21:01:43.734827 INFO::Creating boxplot for categorical data, dysbiosisCD vs Paraprevotella.clara
## 2023-10-24 21:01:43.988463 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.siraeum
## 2023-10-24 21:01:44.249337 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.merdae
## 2023-10-24 21:01:44.517818 INFO::Creating boxplot for categorical data, dysbiosisCD vs Paraprevotella.unclassified
## 2023-10-24 21:01:44.776476 INFO::Creating boxplot for categorical data, dysbiosisCD vs Collinsella.aerofaciens
## 2023-10-24 21:01:45.065123 INFO::Creating boxplot for categorical data, dysbiosisCD vs Odoribacter.splanchnicus
## 2023-10-24 21:01:45.346548 INFO::Creating boxplot for categorical data, dysbiosisCD vs Clostridium.clostridioforme
## 2023-10-24 21:01:45.6591 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.eggerthii
## 2023-10-24 21:01:45.968827 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.onderdonkii
## 2023-10-24 21:01:46.258849 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.ventriosum
## 2023-10-24 21:01:46.586739 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.lactaris
## 2023-10-24 21:01:46.88941 INFO::Creating boxplot for categorical data, dysbiosisCD vs Burkholderiales.bacterium.1.1.47
## 2023-10-24 21:01:47.184024 INFO::Creating boxplot for categorical data, dysbiosisCD vs Dialister.invisus
## 2023-10-24 21:01:47.52132 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.bromii
## 2023-10-24 21:01:47.910208 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parasutterella.excrementihominis
## 2023-10-24 21:01:48.208936 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.unclassified
## 2023-10-24 21:01:48.512332 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.torques
## 2023-10-24 21:01:48.854771 INFO::Creating boxplot for categorical data, dysbiosisCD vs Coprobacillus.unclassified
## 2023-10-24 21:01:49.213485 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.cellulosilyticus
## 2023-10-24 21:01:49.48221 INFO::Creating boxplot for categorical data, dysbiosisCD vs Roseburia.intestinalis
## 2023-10-24 21:01:49.80613 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.unclassified
## 2023-10-24 21:01:50.161019 INFO::Creating boxplot for categorical data, dysbiosisCD vs Acidaminococcus.unclassified
## 2023-10-24 21:01:50.472368 INFO::Creating boxplot for categorical data, dysbiosisCD vs Barnesiella.intestinihominis
## 2023-10-24 21:01:50.745448 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.massiliensis
## 2023-10-24 21:01:51.020162 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.sp.3.1.31
## 2023-10-24 21:01:51.453618 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.salyersiae
## 2023-10-24 21:01:51.732614 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroidales.bacterium.ph8
## 2023-10-24 21:01:52.06405 INFO::Creating boxplot for categorical data, dysbiosisCD vs Clostridium.citroniae
## 2023-10-24 21:01:52.360397 INFO::Creating boxplot for categorical data, dysbiosisCD vs Flavonifractor.plautii
## 2023-10-24 21:01:52.70651 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.goldsteinii
## 2023-10-24 21:01:52.995793 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bifidobacterium.longum
## 2023-10-24 21:01:53.280621 INFO::Creating boxplot for categorical data, dysbiosisCD vs Lachnospiraceae.bacterium.1.1.57FAA
## 2023-10-24 21:01:57.021584 INFO::Plotting data for metadata number 2, dysbiosisUC
## 2023-10-24 21:01:57.025179 INFO::Creating boxplot for categorical data, dysbiosisUC vs Subdoligranulum.unclassified
## 2023-10-24 21:01:57.305151 INFO::Creating boxplot for categorical data, dysbiosisUC vs Faecalibacterium.prausnitzii
## 2023-10-24 21:01:57.689289 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.caccae
## 2023-10-24 21:01:57.992913 INFO::Creating boxplot for categorical data, dysbiosisUC vs Oscillibacter.unclassified
## 2023-10-24 21:01:58.277463 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.uniformis
## 2023-10-24 21:01:58.576575 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.siraeum
## 2023-10-24 21:01:58.855378 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.ovatus
## 2023-10-24 21:01:59.139211 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.fragilis
## 2023-10-24 21:01:59.437533 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.shahii
## 2023-10-24 21:01:59.792673 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.rectale
## 2023-10-24 21:02:00.27007 INFO::Creating boxplot for categorical data, dysbiosisUC vs Roseburia.hominis
## 2023-10-24 21:02:00.538538 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.putredinis
## 2023-10-24 21:02:00.977108 INFO::Creating boxplot for categorical data, dysbiosisUC vs Lachnospiraceae.bacterium.3.1.46FAA
## 2023-10-24 21:02:01.363364 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.hallii
## 2023-10-24 21:02:01.670545 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.xylanisolvens
## 2023-10-24 21:02:02.056114 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.stercoris
## 2023-10-24 21:02:02.340282 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.finegoldii
## 2023-10-24 21:02:02.612841 INFO::Creating boxplot for categorical data, dysbiosisUC vs Barnesiella.intestinihominis
## 2023-10-24 21:02:02.92445 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.leptum
## 2023-10-24 21:02:03.25618 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.gnavus
## 2023-10-24 21:02:03.525149 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.onderdonkii
## 2023-10-24 21:02:03.815333 INFO::Creating boxplot for categorical data, dysbiosisUC vs Flavonifractor.plautii
## 2023-10-24 21:02:04.078605 INFO::Creating boxplot for categorical data, dysbiosisUC vs Parabacteroides.merdae
## 2023-10-24 21:02:04.36276 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.cellulosilyticus
## 2023-10-24 21:02:04.668507 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bifidobacterium.longum
## 2023-10-24 21:02:04.94412 INFO::Creating boxplot for categorical data, dysbiosisUC vs Klebsiella.pneumoniae
## 2023-10-24 21:02:05.244627 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.citroniae
## 2023-10-24 21:02:05.540538 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.ventriosum
## 2023-10-24 21:02:05.852175 INFO::Creating boxplot for categorical data, dysbiosisUC vs Parabacteroides.distasonis
## 2023-10-24 21:02:06.110394 INFO::Creating boxplot for categorical data, dysbiosisUC vs Parabacteroides.goldsteinii
## 2023-10-24 21:02:06.39106 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.torques
## 2023-10-24 21:02:06.69376 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroidales.bacterium.ph8
## 2023-10-24 21:02:07.016534 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.obeum
## 2023-10-24 21:02:07.29459 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bifidobacterium.bifidum
## 2023-10-24 21:02:07.577177 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bifidobacterium.adolescentis
## 2023-10-24 21:02:07.851071 INFO::Creating boxplot for categorical data, dysbiosisUC vs Collinsella.aerofaciens
## 2023-10-24 21:02:08.133705 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.hathewayi
## 2023-10-24 21:02:08.39697 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bilophila.unclassified
## 2023-10-24 21:02:08.718671 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.eligens
## 2023-10-24 21:02:09.015335 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.vulgatus
## 2023-10-24 21:02:09.282189 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.bolteae
## 2023-10-24 21:02:09.573275 INFO::Creating boxplot for categorical data, dysbiosisUC vs Dialister.invisus
## 2023-10-24 21:02:09.87581 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.lactaris
## 2023-10-24 21:02:10.140309 INFO::Creating boxplot for categorical data, dysbiosisUC vs Burkholderiales.bacterium.1.1.47
## 2023-10-24 21:02:14.21904 INFO::Plotting data for metadata number 3, dysbiosisnonIBD
## 2023-10-24 21:02:14.22231 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Faecalibacterium.prausnitzii
## 2023-10-24 21:02:14.461279 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.rectale
## 2023-10-24 21:02:14.783157 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.sp.3.1.31
## 2023-10-24 21:02:15.086814 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.hominis
## 2023-10-24 21:02:15.364916 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Subdoligranulum.unclassified
## 2023-10-24 21:02:15.685622 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Lachnospiraceae.bacterium.3.1.46FAA
## 2023-10-24 21:02:15.957519 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Ruminococcus.torques
## 2023-10-24 21:02:16.252297 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Clostridium.leptum
## 2023-10-24 21:02:16.523613 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Odoribacter.splanchnicus
## 2023-10-24 21:02:16.847163 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Escherichia.coli
## 2023-10-24 21:02:17.1436 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Ruminococcus.obeum
## 2023-10-24 21:02:17.412244 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bilophila.unclassified
## 2023-10-24 21:02:17.752511 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.uniformis
## 2023-10-24 21:02:18.103948 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.eligens
## 2023-10-24 21:02:18.579791 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Klebsiella.pneumoniae
## 2023-10-24 21:02:18.915357 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Ruminococcus.bromii
## 2023-10-24 21:02:19.212414 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.vulgatus
## 2023-10-24 21:02:19.508388 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs C2likevirus.unclassified
## 2023-10-24 21:02:19.874144 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bifidobacterium.adolescentis
## 2023-10-24 21:02:20.149501 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Alistipes.finegoldii
## 2023-10-24 21:02:20.446944 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.inulinivorans
## 2023-10-24 21:02:20.750548 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Alistipes.onderdonkii
## 2023-10-24 21:02:21.048534 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.unclassified
## 2023-10-24 21:02:21.361115 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.hallii
## 2023-10-24 21:02:21.677271 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.intestinalis
## 2023-10-24 21:02:21.976383 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Prevotella.copri
## 2023-10-24 21:02:22.324406 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.dorei
## 2023-10-24 21:02:22.618655 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.fragilis
## 2023-10-24 21:02:22.880455 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Alistipes.shahii
## 2023-10-24 21:02:27.0791 INFO::Plotting data for metadata number 4, antibiotics
## 2023-10-24 21:02:27.082948 INFO::Creating boxplot for categorical data, antibiotics vs Roseburia.inulinivorans
## 2023-10-24 21:02:27.318616 INFO::Creating boxplot for categorical data, antibiotics vs Roseburia.hominis
## 2023-10-24 21:02:27.636338 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.rectale
## 2023-10-24 21:02:27.894538 INFO::Creating boxplot for categorical data, antibiotics vs Dialister.invisus
## 2023-10-24 21:02:28.191479 INFO::Creating boxplot for categorical data, antibiotics vs Roseburia.intestinalis
## 2023-10-24 21:02:28.467062 INFO::Creating boxplot for categorical data, antibiotics vs Dorea.longicatena
## 2023-10-24 21:02:28.806108 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.callidus
## 2023-10-24 21:02:29.115749 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.bromii
## 2023-10-24 21:02:29.378214 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.obeum
## 2023-10-24 21:02:29.676044 INFO::Creating boxplot for categorical data, antibiotics vs Klebsiella.pneumoniae
## 2023-10-24 21:02:29.935112 INFO::Creating boxplot for categorical data, antibiotics vs Bifidobacterium.adolescentis
## 2023-10-24 21:02:30.210787 INFO::Creating boxplot for categorical data, antibiotics vs Faecalibacterium.prausnitzii
## 2023-10-24 21:02:30.460274 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.hallii
## 2023-10-24 21:02:30.764657 INFO::Creating boxplot for categorical data, antibiotics vs Bilophila.unclassified
## 2023-10-24 21:02:31.05194 INFO::Creating boxplot for categorical data, antibiotics vs Clostridium.leptum
## 2023-10-24 21:02:31.312049 INFO::Creating boxplot for categorical data, antibiotics vs Lachnospiraceae.bacterium.3.1.46FAA
## 2023-10-24 21:02:31.60855 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.finegoldii
## 2023-10-24 21:02:31.935899 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.sp.3.1.31
## 2023-10-24 21:02:32.224925 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.fragilis
## 2023-10-24 21:02:32.50833 INFO::Creating boxplot for categorical data, antibiotics vs Alistipes.onderdonkii
## 2023-10-24 21:02:32.790352 INFO::Creating boxplot for categorical data, antibiotics vs Sutterella.wadsworthensis
## 2023-10-24 21:02:33.086382 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.eligens
## 2023-10-24 21:02:33.360641 INFO::Creating boxplot for categorical data, antibiotics vs Collinsella.aerofaciens
## 2023-10-24 21:02:33.658571 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.thetaiotaomicron
## 2023-10-24 21:02:33.943612 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.eggerthii
## 2023-10-24 21:02:34.219633 INFO::Creating boxplot for categorical data, antibiotics vs Haemophilus.parainfluenzae
## 2023-10-24 21:02:34.487802 INFO::Creating boxplot for categorical data, antibiotics vs Bifidobacterium.pseudocatenulatum
## 2023-10-24 21:02:34.792372 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.torques
## 2023-10-24 21:02:35.031244 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.ventriosum
## 2023-10-24 21:02:35.322853 INFO::Creating boxplot for categorical data, antibiotics vs Parasutterella.excrementihominis
## 2023-10-24 21:02:35.913712 INFO::Creating boxplot for categorical data, antibiotics vs Peptostreptococcaceae.noname.unclassified
## 2023-10-24 21:02:36.148449 INFO::Creating boxplot for categorical data, antibiotics vs Veillonella.dispar
## 2023-10-24 21:02:36.387551 INFO::Creating boxplot for categorical data, antibiotics vs Veillonella.atypica
## 2023-10-24 21:02:36.629643 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroidales.bacterium.ph8
## 2023-10-24 21:02:36.871192 INFO::Creating boxplot for categorical data, antibiotics vs Clostridium.nexile
## 2023-10-24 21:02:37.101842 INFO::Creating boxplot for categorical data, antibiotics vs Burkholderiales.bacterium.1.1.47
## 2023-10-24 21:02:37.33597 INFO::Creating boxplot for categorical data, antibiotics vs Lachnospiraceae.bacterium.1.1.57FAA
## 2023-10-24 21:02:37.56698 INFO::Creating boxplot for categorical data, antibiotics vs Akkermansia.muciniphila
## 2023-10-24 21:02:37.813686 INFO::Creating boxplot for categorical data, antibiotics vs Clostridium.citroniae
## 2023-10-24 21:02:38.043314 INFO::Creating boxplot for categorical data, antibiotics vs Odoribacter.splanchnicus
## 2023-10-24 21:02:41.273976 INFO::Plotting data for metadata number 5, age
## 2023-10-24 21:02:41.277074 INFO::Creating scatter plot for continuous data, age vs Haemophilus.parainfluenzae
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:41.559263 INFO::Creating scatter plot for continuous data, age vs Bifidobacterium.pseudocatenulatum
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:41.903198 INFO::Creating scatter plot for continuous data, age vs Faecalibacterium.prausnitzii
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:42.144094 INFO::Creating scatter plot for continuous data, age vs Clostridium.clostridioforme
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:42.411926 INFO::Creating scatter plot for continuous data, age vs Veillonella.parvula
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:42.668709 INFO::Creating scatter plot for continuous data, age vs Subdoligranulum.unclassified
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:42.941385 INFO::Creating scatter plot for continuous data, age vs Clostridium.symbiosum
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:43.210679 INFO::Creating scatter plot for continuous data, age vs Ruminococcus.gnavus
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:43.473353 INFO::Creating scatter plot for continuous data, age vs Dialister.invisus
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:43.733443 INFO::Creating scatter plot for continuous data, age vs Veillonella.dispar
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:43.994672 INFO::Creating scatter plot for continuous data, age vs Veillonella.unclassified
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:44.237115 INFO::Creating scatter plot for continuous data, age vs Bacteroides.thetaiotaomicron
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:44.499588 INFO::Creating scatter plot for continuous data, age vs Ruminococcus.bromii
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:44.820491 INFO::Creating scatter plot for continuous data, age vs Bacteroides.intestinalis
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:45.136062 INFO::Creating scatter plot for continuous data, age vs Eubacterium.siraeum
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:45.387023 INFO::Creating scatter plot for continuous data, age vs Prevotella.copri
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:45.662806 INFO::Creating scatter plot for continuous data, age vs Alistipes.unclassified
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:45.922031 INFO::Creating scatter plot for continuous data, age vs Bacteroidales.bacterium.ph8
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:46.174348 INFO::Creating scatter plot for continuous data, age vs Bifidobacterium.longum
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:46.443023 INFO::Creating scatter plot for continuous data, age vs Akkermansia.muciniphila
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:46.740647 INFO::Creating scatter plot for continuous data, age vs Collinsella.aerofaciens
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:47.006858 INFO::Creating scatter plot for continuous data, age vs Parabacteroides.distasonis
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## `geom_smooth()` using formula = 'y ~ x'
## Warning: Removed 19 rows containing missing values (`geom_point()`).
## 2023-10-24 21:02:50.723578 INFO::Plotting data for metadata number 6, diagnosis
## 2023-10-24 21:02:50.726743 INFO::Creating boxplot for categorical data, diagnosis vs Bifidobacterium.adolescentis
## 2023-10-24 21:02:50.952249 INFO::Creating boxplot for categorical data, diagnosis vs Akkermansia.muciniphila
## 2023-10-24 21:02:51.219304 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.bolteae
## 2023-10-24 21:02:51.487283 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.putredinis
## 2023-10-24 21:02:51.783793 INFO::Creating boxplot for categorical data, diagnosis vs Coprobacillus.unclassified
## 2023-10-24 21:02:52.050852 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.clostridioforme
## 2023-10-24 21:02:52.293663 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.shahii
## 2023-10-24 21:02:52.55471 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.bromii
## 2023-10-24 21:02:52.86837 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.leptum
## 2023-10-24 21:02:53.143521 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.symbiosum
## 2023-10-24 21:02:53.401074 INFO::Creating boxplot for categorical data, diagnosis vs Roseburia.inulinivorans
## 2023-10-24 21:02:53.666839 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.bromii
## 2023-10-24 21:02:53.935032 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.shahii
## 2023-10-24 21:02:54.182261 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.finegoldii
## 2023-10-24 21:02:54.530843 INFO::Creating boxplot for categorical data, diagnosis vs Parabacteroides.goldsteinii
## 2023-10-24 21:02:54.877708 INFO::Creating boxplot for categorical data, diagnosis vs Bilophila.unclassified
## 2023-10-24 21:02:55.149112 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.leptum
## 2023-10-24 21:02:55.526782 INFO::Creating boxplot for categorical data, diagnosis vs Roseburia.hominis
## 2023-10-24 21:02:55.836375 INFO::Creating boxplot for categorical data, diagnosis vs Coprobacillus.unclassified
## 2023-10-24 21:02:56.122541 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.onderdonkii
## 2023-10-24 21:02:56.412029 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.ventriosum
## 2023-10-24 21:02:56.726862 INFO::Creating boxplot for categorical data, diagnosis vs Roseburia.hominis
## 2023-10-24 21:02:57.057508 INFO::Creating boxplot for categorical data, diagnosis vs Sutterella.wadsworthensis
## 2023-10-24 21:02:57.333681 INFO::Creating boxplot for categorical data, diagnosis vs Akkermansia.muciniphila
## 2023-10-24 21:02:57.606441 INFO::Creating boxplot for categorical data, diagnosis vs Sutterella.wadsworthensis
## 2023-10-24 21:02:57.926223 INFO::Creating boxplot for categorical data, diagnosis vs Subdoligranulum.unclassified
## 2023-10-24 21:02:58.191576 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.symbiosum
## 2023-10-24 21:02:58.515957 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.onderdonkii
## 2023-10-24 21:02:58.859371 INFO::Creating boxplot for categorical data, diagnosis vs Parabacteroides.unclassified
## 2023-10-24 21:02:59.132745 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.lactaris
## 2023-10-24 21:02:59.425648 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.callidus
## 2023-10-24 21:02:59.733234 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.gnavus
## 2023-10-24 21:02:59.985659 INFO::Creating boxplot for categorical data, diagnosis vs Bacteroides.fragilis
## 2023-10-24 21:03:00.270922 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.rectale
## 2023-10-24 21:03:00.549491 INFO::Creating boxplot for categorical data, diagnosis vs Odoribacter.splanchnicus
## 2023-10-24 21:03:00.871821 INFO::Creating boxplot for categorical data, diagnosis vs Parabacteroides.distasonis
## 2023-10-24 21:03:01.154788 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.rectale
## 2023-10-24 21:03:01.442058 INFO::Creating boxplot for categorical data, diagnosis vs Bacteroides.finegoldii
## 2023-10-24 21:03:01.767345 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.callidus
## 2023-10-24 21:03:02.033829 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.finegoldii
## 2023-10-24 21:03:02.310595 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.putredinis
## 2023-10-24 21:03:02.594596 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.bolteae
## 2023-10-24 21:03:02.85744 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.siraeum
Session Info

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

sessionInfo()
## R version 4.3.1 (2023-06-16 ucrt)
## Platform: x86_64-w64-mingw32/x64 (64-bit)
## 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_1.16.0
## 
## loaded via a namespace (and not attached):
##  [1] gtable_0.3.4        biglm_0.9-2.1       xfun_0.40          
##  [4] bslib_0.5.1         ggplot2_3.4.4       lattice_0.22-5     
##  [7] numDeriv_2016.8-1.1 vctrs_0.6.4         tools_4.3.1        
## [10] generics_0.1.3      parallel_4.3.1      getopt_1.20.4      
## [13] tibble_3.2.1        fansi_1.0.5         DEoptimR_1.1-3     
## [16] cluster_2.1.4       pkgconfig_2.0.3     logging_0.10-108   
## [19] pheatmap_1.0.12     Matrix_1.6-1.1      data.table_1.14.8  
## [22] RColorBrewer_1.1-3  lifecycle_1.0.3     farver_2.1.1       
## [25] compiler_4.3.1      munsell_0.5.0       lmerTest_3.1-3     
## [28] permute_0.9-7       htmltools_0.5.6.1   sass_0.4.7         
## [31] hash_2.2.6.3        yaml_2.3.7          pillar_1.9.0       
## [34] nloptr_2.0.3        crayon_1.5.2        jquerylib_0.1.4    
## [37] MASS_7.3-60         cachem_1.0.8        vegan_2.6-4        
## [40] boot_1.3-28.1       nlme_3.1-163        robustbase_0.99-0  
## [43] tidyselect_1.2.0    digest_0.6.33       mvtnorm_1.2-3      
## [46] dplyr_1.1.3         labeling_0.4.3      splines_4.3.1      
## [49] pcaPP_2.0-3         fastmap_1.1.1       grid_4.3.1         
## [52] colorspace_2.1-0    cli_3.6.1           magrittr_2.0.3     
## [55] utf8_1.2.4          withr_2.5.1         scales_1.2.1       
## [58] rmarkdown_2.25      lme4_1.1-34         pbapply_1.7-2      
## [61] evaluate_0.22       knitr_1.44          mgcv_1.9-0         
## [64] rlang_1.1.1         Rcpp_1.0.11         glue_1.6.2         
## [67] optparse_1.7.3      DBI_1.1.3           minqa_1.2.6        
## [70] jsonlite_1.8.7      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.