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 17:36:37.866638 INFO::Writing function arguments to log file
## 2023-10-24 17:36:37.900388 INFO::Verifying options selected are valid
## 2023-10-24 17:36:37.939827 INFO::Determining format of input files
## 2023-10-24 17:36:37.941468 INFO::Input format is data samples as rows and metadata samples as rows
## 2023-10-24 17:36:37.948388 INFO::Formula for random effects: expr ~ (1 | site) + (1 | subject)
## 2023-10-24 17:36:37.950245 INFO::Formula for fixed effects: expr ~  diagnosis + dysbiosisnonIBD + dysbiosisUC + dysbiosisCD + antibiotics + age
## 2023-10-24 17:36:37.953738 INFO::Filter data based on min abundance and min prevalence
## 2023-10-24 17:36:37.954554 INFO::Total samples in data: 1595
## 2023-10-24 17:36:37.95553 INFO::Min samples required with min abundance for a feature not to be filtered: 159.500000
## 2023-10-24 17:36:37.961312 INFO::Total filtered features: 0
## 2023-10-24 17:36:37.962503 INFO::Filtered feature names from abundance and prevalence filtering:
## 2023-10-24 17:36:37.972447 INFO::Total filtered features with variance filtering: 0
## 2023-10-24 17:36:37.973623 INFO::Filtered feature names from variance filtering:
## 2023-10-24 17:36:37.974437 INFO::Running selected normalization method: TSS
## 2023-10-24 17:36:39.272847 INFO::Bypass z-score application to metadata
## 2023-10-24 17:36:39.274302 INFO::Running selected transform method: LOG
## 2023-10-24 17:36:39.315936 INFO::Running selected analysis method: LM
## 2023-10-24 17:36:39.933004 INFO::Fitting model to feature number 1, Bifidobacterium.adolescentis
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:40.335963 INFO::Fitting model to feature number 2, Bifidobacterium.bifidum
## 2023-10-24 17:36:40.551479 INFO::Fitting model to feature number 3, Bifidobacterium.longum
## 2023-10-24 17:36:40.75244 INFO::Fitting model to feature number 4, Bifidobacterium.pseudocatenulatum
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:40.92462 INFO::Fitting model to feature number 5, Collinsella.aerofaciens
## 2023-10-24 17:36:41.117922 INFO::Fitting model to feature number 6, Bacteroides.caccae
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:41.296265 INFO::Fitting model to feature number 7, Bacteroides.cellulosilyticus
## 2023-10-24 17:36:41.465654 INFO::Fitting model to feature number 8, Bacteroides.dorei
## 2023-10-24 17:36:41.637611 INFO::Fitting model to feature number 9, Bacteroides.eggerthii
## 2023-10-24 17:36:41.801903 INFO::Fitting model to feature number 10, Bacteroides.faecis
## 2023-10-24 17:36:41.992842 INFO::Fitting model to feature number 11, Bacteroides.finegoldii
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:42.154729 INFO::Fitting model to feature number 12, Bacteroides.fragilis
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:42.300428 INFO::Fitting model to feature number 13, Bacteroides.intestinalis
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:42.478599 INFO::Fitting model to feature number 14, Bacteroides.massiliensis
## 2023-10-24 17:36:42.632187 INFO::Fitting model to feature number 15, Bacteroides.ovatus
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:42.797738 INFO::Fitting model to feature number 16, Bacteroides.salyersiae
## 2023-10-24 17:36:42.969254 INFO::Fitting model to feature number 17, Bacteroides.stercoris
## 2023-10-24 17:36:43.147828 INFO::Fitting model to feature number 18, Bacteroides.thetaiotaomicron
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:43.318409 INFO::Fitting model to feature number 19, Bacteroides.uniformis
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:43.499713 INFO::Fitting model to feature number 20, Bacteroides.vulgatus
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:43.681566 INFO::Fitting model to feature number 21, Bacteroides.xylanisolvens
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:43.860192 INFO::Fitting model to feature number 22, Bacteroidales.bacterium.ph8
## 2023-10-24 17:36:44.033528 INFO::Fitting model to feature number 23, Barnesiella.intestinihominis
## 2023-10-24 17:36:44.238787 INFO::Fitting model to feature number 24, Coprobacter.fastidiosus
## 2023-10-24 17:36:44.421158 INFO::Fitting model to feature number 25, Odoribacter.splanchnicus
## 2023-10-24 17:36:44.605526 INFO::Fitting model to feature number 26, Parabacteroides.distasonis
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:45.008954 INFO::Fitting model to feature number 27, Parabacteroides.goldsteinii
## 2023-10-24 17:36:45.167724 INFO::Fitting model to feature number 28, Parabacteroides.merdae
## 2023-10-24 17:36:45.326404 INFO::Fitting model to feature number 29, Parabacteroides.unclassified
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:45.497803 INFO::Fitting model to feature number 30, Paraprevotella.clara
## 2023-10-24 17:36:45.662095 INFO::Fitting model to feature number 31, Paraprevotella.unclassified
## 2023-10-24 17:36:45.820829 INFO::Fitting model to feature number 32, Prevotella.copri
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:45.97328 INFO::Fitting model to feature number 33, Alistipes.finegoldii
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:46.146481 INFO::Fitting model to feature number 34, Alistipes.onderdonkii
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:46.312592 INFO::Fitting model to feature number 35, Alistipes.putredinis
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:46.460526 INFO::Fitting model to feature number 36, Alistipes.shahii
## 2023-10-24 17:36:46.844023 INFO::Fitting model to feature number 37, Alistipes.unclassified
## 2023-10-24 17:36:46.989249 INFO::Fitting model to feature number 38, Streptococcus.salivarius
## 2023-10-24 17:36:47.136778 INFO::Fitting model to feature number 39, Clostridium.bolteae
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:47.266683 INFO::Fitting model to feature number 40, Clostridium.citroniae
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:47.405844 INFO::Fitting model to feature number 41, Clostridium.clostridioforme
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:47.528307 INFO::Fitting model to feature number 42, Clostridium.hathewayi
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:47.674428 INFO::Fitting model to feature number 43, Clostridium.leptum
## 2023-10-24 17:36:47.855225 INFO::Fitting model to feature number 44, Clostridium.nexile
## 2023-10-24 17:36:48.022162 INFO::Fitting model to feature number 45, Clostridium.symbiosum
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:48.179847 INFO::Fitting model to feature number 46, Flavonifractor.plautii
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:48.339642 INFO::Fitting model to feature number 47, Eubacterium.eligens
## 2023-10-24 17:36:48.495369 INFO::Fitting model to feature number 48, Eubacterium.hallii
## 2023-10-24 17:36:48.650883 INFO::Fitting model to feature number 49, Eubacterium.rectale
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:48.805985 INFO::Fitting model to feature number 50, Eubacterium.siraeum
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:48.935178 INFO::Fitting model to feature number 51, Eubacterium.sp.3.1.31
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:49.093808 INFO::Fitting model to feature number 52, Eubacterium.ventriosum
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:49.23014 INFO::Fitting model to feature number 53, Ruminococcus.gnavus
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:49.395514 INFO::Fitting model to feature number 54, Ruminococcus.obeum
## 2023-10-24 17:36:49.536023 INFO::Fitting model to feature number 55, Ruminococcus.torques
## 2023-10-24 17:36:49.65855 INFO::Fitting model to feature number 56, Coprococcus.comes
## 2023-10-24 17:36:49.828191 INFO::Fitting model to feature number 57, Dorea.longicatena
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:49.985283 INFO::Fitting model to feature number 58, Lachnospiraceae.bacterium.1.1.57FAA
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:50.139504 INFO::Fitting model to feature number 59, Lachnospiraceae.bacterium.3.1.46FAA
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:50.532436 INFO::Fitting model to feature number 60, Roseburia.hominis
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:50.685117 INFO::Fitting model to feature number 61, Roseburia.intestinalis
## 2023-10-24 17:36:50.842513 INFO::Fitting model to feature number 62, Roseburia.inulinivorans
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:51.237747 INFO::Fitting model to feature number 63, Roseburia.unclassified
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:51.386628 INFO::Fitting model to feature number 64, Oscillibacter.unclassified
## 2023-10-24 17:36:51.563956 INFO::Fitting model to feature number 65, Peptostreptococcaceae.noname.unclassified
## 2023-10-24 17:36:51.717513 INFO::Fitting model to feature number 66, Faecalibacterium.prausnitzii
## 2023-10-24 17:36:51.859088 INFO::Fitting model to feature number 67, Ruminococcus.bromii
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:51.995544 INFO::Fitting model to feature number 68, Ruminococcus.callidus
## 2023-10-24 17:36:52.140552 INFO::Fitting model to feature number 69, Ruminococcus.lactaris
## 2023-10-24 17:36:52.310423 INFO::Fitting model to feature number 70, Subdoligranulum.unclassified
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:52.463246 INFO::Fitting model to feature number 71, Coprobacillus.unclassified
## 2023-10-24 17:36:52.611144 INFO::Fitting model to feature number 72, Acidaminococcus.unclassified
## 2023-10-24 17:36:52.798594 INFO::Fitting model to feature number 73, Dialister.invisus
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:52.961885 INFO::Fitting model to feature number 74, Veillonella.atypica
## 2023-10-24 17:36:53.12278 INFO::Fitting model to feature number 75, Veillonella.dispar
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:53.295718 INFO::Fitting model to feature number 76, Veillonella.parvula
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:53.459286 INFO::Fitting model to feature number 77, Veillonella.unclassified
## 2023-10-24 17:36:53.630167 INFO::Fitting model to feature number 78, Burkholderiales.bacterium.1.1.47
## 2023-10-24 17:36:53.816929 INFO::Fitting model to feature number 79, Parasutterella.excrementihominis
## 2023-10-24 17:36:53.981606 INFO::Fitting model to feature number 80, Sutterella.wadsworthensis
## 2023-10-24 17:36:54.164006 INFO::Fitting model to feature number 81, Bilophila.unclassified
## 2023-10-24 17:36:54.330953 INFO::Fitting model to feature number 82, Escherichia.coli
## 2023-10-24 17:36:54.503061 INFO::Fitting model to feature number 83, Escherichia.unclassified
## 2023-10-24 17:36:54.664118 INFO::Fitting model to feature number 84, Klebsiella.pneumoniae
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:54.826668 INFO::Fitting model to feature number 85, Haemophilus.parainfluenzae
## boundary (singular) fit: see help('isSingular')
## 2023-10-24 17:36:54.991564 INFO::Fitting model to feature number 86, Akkermansia.muciniphila
## 2023-10-24 17:36:55.167429 INFO::Fitting model to feature number 87, C2likevirus.unclassified
## 2023-10-24 17:36:55.384919 INFO::Counting total values for each feature
## 2023-10-24 17:36:55.4335 INFO::Writing filtered data to file demo_output/features/filtered_data.tsv
## 2023-10-24 17:36:55.569312 INFO::Writing filtered, normalized data to file demo_output/features/filtered_data_norm.tsv
## 2023-10-24 17:36:55.725225 INFO::Writing filtered, normalized, transformed data to file demo_output/features/filtered_data_norm_transformed.tsv
## 2023-10-24 17:36:55.951421 INFO::Writing residuals to file demo_output/fits/residuals.rds
## 2023-10-24 17:36:56.018881 INFO::Writing fitted values to file demo_output/fits/fitted.rds
## 2023-10-24 17:36:56.05018 INFO::Writing extracted random effects to file demo_output/fits/ranef.rds
## 2023-10-24 17:36:56.057545 INFO::Writing all results to file (ordered by increasing q-values): demo_output/all_results.tsv
## 2023-10-24 17:36:56.065872 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 17:36:56.070659 INFO::Writing heatmap of significant results to file: demo_output/heatmap.pdf
## 2023-10-24 17:36:56.341331 INFO::Writing association plots (one for each significant association) to output folder: demo_output
## 2023-10-24 17:36:56.347991 INFO::Plotting associations from most to least significant, grouped by metadata
## 2023-10-24 17:36:56.349528 INFO::Plotting data for metadata number 1, dysbiosisCD
## 2023-10-24 17:36:56.351965 INFO::Creating boxplot for categorical data, dysbiosisCD vs Faecalibacterium.prausnitzii
## 2023-10-24 17:36:56.958659 INFO::Creating boxplot for categorical data, dysbiosisCD vs Subdoligranulum.unclassified
## 2023-10-24 17:36:57.247032 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.uniformis
## 2023-10-24 17:36:57.518469 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.rectale
## 2023-10-24 17:36:57.822301 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.vulgatus
## 2023-10-24 17:36:58.101516 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.ovatus
## 2023-10-24 17:36:58.424868 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.obeum
## 2023-10-24 17:36:58.695413 INFO::Creating boxplot for categorical data, dysbiosisCD vs Oscillibacter.unclassified
## 2023-10-24 17:36:58.989584 INFO::Creating boxplot for categorical data, dysbiosisCD vs Roseburia.inulinivorans
## 2023-10-24 17:36:59.288647 INFO::Creating boxplot for categorical data, dysbiosisCD vs Roseburia.hominis
## 2023-10-24 17:36:59.598448 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.thetaiotaomicron
## 2023-10-24 17:36:59.955275 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.putredinis
## 2023-10-24 17:37:00.384752 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.distasonis
## 2023-10-24 17:37:00.732488 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.dorei
## 2023-10-24 17:37:01.078705 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.shahii
## 2023-10-24 17:37:01.4041 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.xylanisolvens
## 2023-10-24 17:37:01.782826 INFO::Creating boxplot for categorical data, dysbiosisCD vs Clostridium.leptum
## 2023-10-24 17:37:02.167249 INFO::Creating boxplot for categorical data, dysbiosisCD vs Dorea.longicatena
## 2023-10-24 17:37:02.575511 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.caccae
## 2023-10-24 17:37:02.950183 INFO::Creating boxplot for categorical data, dysbiosisCD vs Lachnospiraceae.bacterium.3.1.46FAA
## 2023-10-24 17:37:03.332923 INFO::Creating boxplot for categorical data, dysbiosisCD vs Escherichia.coli
## 2023-10-24 17:37:03.731287 INFO::Creating boxplot for categorical data, dysbiosisCD vs Klebsiella.pneumoniae
## 2023-10-24 17:37:04.15271 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bilophila.unclassified
## 2023-10-24 17:37:04.504563 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.finegoldii
## 2023-10-24 17:37:04.837776 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.eligens
## 2023-10-24 17:37:05.25347 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.stercoris
## 2023-10-24 17:37:05.598785 INFO::Creating boxplot for categorical data, dysbiosisCD vs Coprococcus.comes
## 2023-10-24 17:37:06.034376 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.fragilis
## 2023-10-24 17:37:06.386773 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.finegoldii
## 2023-10-24 17:37:06.794549 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.hallii
## 2023-10-24 17:37:07.105545 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.faecis
## 2023-10-24 17:37:07.395434 INFO::Creating boxplot for categorical data, dysbiosisCD vs Paraprevotella.clara
## 2023-10-24 17:37:07.681319 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.siraeum
## 2023-10-24 17:37:07.969996 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.merdae
## 2023-10-24 17:37:08.24664 INFO::Creating boxplot for categorical data, dysbiosisCD vs Paraprevotella.unclassified
## 2023-10-24 17:37:08.623845 INFO::Creating boxplot for categorical data, dysbiosisCD vs Collinsella.aerofaciens
## 2023-10-24 17:37:08.932705 INFO::Creating boxplot for categorical data, dysbiosisCD vs Odoribacter.splanchnicus
## 2023-10-24 17:37:09.223429 INFO::Creating boxplot for categorical data, dysbiosisCD vs Clostridium.clostridioforme
## 2023-10-24 17:37:09.528949 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.eggerthii
## 2023-10-24 17:37:09.799828 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.onderdonkii
## 2023-10-24 17:37:10.10425 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.ventriosum
## 2023-10-24 17:37:10.367357 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.lactaris
## 2023-10-24 17:37:10.668689 INFO::Creating boxplot for categorical data, dysbiosisCD vs Burkholderiales.bacterium.1.1.47
## 2023-10-24 17:37:10.943528 INFO::Creating boxplot for categorical data, dysbiosisCD vs Dialister.invisus
## 2023-10-24 17:37:11.224638 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.bromii
## 2023-10-24 17:37:11.510627 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parasutterella.excrementihominis
## 2023-10-24 17:37:11.769657 INFO::Creating boxplot for categorical data, dysbiosisCD vs Alistipes.unclassified
## 2023-10-24 17:37:12.050384 INFO::Creating boxplot for categorical data, dysbiosisCD vs Ruminococcus.torques
## 2023-10-24 17:37:12.341872 INFO::Creating boxplot for categorical data, dysbiosisCD vs Coprobacillus.unclassified
## 2023-10-24 17:37:12.617818 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.cellulosilyticus
## 2023-10-24 17:37:12.914313 INFO::Creating boxplot for categorical data, dysbiosisCD vs Roseburia.intestinalis
## 2023-10-24 17:37:13.200878 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.unclassified
## 2023-10-24 17:37:13.498807 INFO::Creating boxplot for categorical data, dysbiosisCD vs Acidaminococcus.unclassified
## 2023-10-24 17:37:13.788005 INFO::Creating boxplot for categorical data, dysbiosisCD vs Barnesiella.intestinihominis
## 2023-10-24 17:37:14.065362 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.massiliensis
## 2023-10-24 17:37:14.33055 INFO::Creating boxplot for categorical data, dysbiosisCD vs Eubacterium.sp.3.1.31
## 2023-10-24 17:37:14.617397 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroides.salyersiae
## 2023-10-24 17:37:14.859831 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bacteroidales.bacterium.ph8
## 2023-10-24 17:37:15.145323 INFO::Creating boxplot for categorical data, dysbiosisCD vs Clostridium.citroniae
## 2023-10-24 17:37:15.435471 INFO::Creating boxplot for categorical data, dysbiosisCD vs Flavonifractor.plautii
## 2023-10-24 17:37:15.698423 INFO::Creating boxplot for categorical data, dysbiosisCD vs Parabacteroides.goldsteinii
## 2023-10-24 17:37:15.982805 INFO::Creating boxplot for categorical data, dysbiosisCD vs Bifidobacterium.longum
## 2023-10-24 17:37:16.255937 INFO::Creating boxplot for categorical data, dysbiosisCD vs Lachnospiraceae.bacterium.1.1.57FAA
## 2023-10-24 17:37:19.787456 INFO::Plotting data for metadata number 2, dysbiosisUC
## 2023-10-24 17:37:19.789906 INFO::Creating boxplot for categorical data, dysbiosisUC vs Subdoligranulum.unclassified
## 2023-10-24 17:37:20.048899 INFO::Creating boxplot for categorical data, dysbiosisUC vs Faecalibacterium.prausnitzii
## 2023-10-24 17:37:20.335472 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.caccae
## 2023-10-24 17:37:20.628135 INFO::Creating boxplot for categorical data, dysbiosisUC vs Oscillibacter.unclassified
## 2023-10-24 17:37:20.926875 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.uniformis
## 2023-10-24 17:37:21.222787 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.siraeum
## 2023-10-24 17:37:21.47615 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.ovatus
## 2023-10-24 17:37:21.734098 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.fragilis
## 2023-10-24 17:37:22.047713 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.shahii
## 2023-10-24 17:37:22.32298 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.rectale
## 2023-10-24 17:37:22.639625 INFO::Creating boxplot for categorical data, dysbiosisUC vs Roseburia.hominis
## 2023-10-24 17:37:22.931604 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.putredinis
## 2023-10-24 17:37:23.245893 INFO::Creating boxplot for categorical data, dysbiosisUC vs Lachnospiraceae.bacterium.3.1.46FAA
## 2023-10-24 17:37:23.567863 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.hallii
## 2023-10-24 17:37:23.865525 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.xylanisolvens
## 2023-10-24 17:37:24.164667 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.stercoris
## 2023-10-24 17:37:24.444842 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.finegoldii
## 2023-10-24 17:37:24.756563 INFO::Creating boxplot for categorical data, dysbiosisUC vs Barnesiella.intestinihominis
## 2023-10-24 17:37:25.084461 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.leptum
## 2023-10-24 17:37:25.415545 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.gnavus
## 2023-10-24 17:37:25.736679 INFO::Creating boxplot for categorical data, dysbiosisUC vs Alistipes.onderdonkii
## 2023-10-24 17:37:26.062463 INFO::Creating boxplot for categorical data, dysbiosisUC vs Flavonifractor.plautii
## 2023-10-24 17:37:26.411475 INFO::Creating boxplot for categorical data, dysbiosisUC vs Parabacteroides.merdae
## 2023-10-24 17:37:26.735592 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.cellulosilyticus
## 2023-10-24 17:37:27.069712 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bifidobacterium.longum
## 2023-10-24 17:37:27.399338 INFO::Creating boxplot for categorical data, dysbiosisUC vs Klebsiella.pneumoniae
## 2023-10-24 17:37:27.727252 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.citroniae
## 2023-10-24 17:37:28.038775 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.ventriosum
## 2023-10-24 17:37:28.377293 INFO::Creating boxplot for categorical data, dysbiosisUC vs Parabacteroides.distasonis
## 2023-10-24 17:37:28.685075 INFO::Creating boxplot for categorical data, dysbiosisUC vs Parabacteroides.goldsteinii
## 2023-10-24 17:37:29.078983 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.torques
## 2023-10-24 17:37:29.384038 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroidales.bacterium.ph8
## 2023-10-24 17:37:29.730766 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.obeum
## 2023-10-24 17:37:30.043745 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bifidobacterium.bifidum
## 2023-10-24 17:37:30.331669 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bifidobacterium.adolescentis
## 2023-10-24 17:37:30.637074 INFO::Creating boxplot for categorical data, dysbiosisUC vs Collinsella.aerofaciens
## 2023-10-24 17:37:30.950611 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.hathewayi
## 2023-10-24 17:37:31.27217 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bilophila.unclassified
## 2023-10-24 17:37:31.596774 INFO::Creating boxplot for categorical data, dysbiosisUC vs Eubacterium.eligens
## 2023-10-24 17:37:31.880652 INFO::Creating boxplot for categorical data, dysbiosisUC vs Bacteroides.vulgatus
## 2023-10-24 17:37:32.163075 INFO::Creating boxplot for categorical data, dysbiosisUC vs Clostridium.bolteae
## 2023-10-24 17:37:32.459839 INFO::Creating boxplot for categorical data, dysbiosisUC vs Dialister.invisus
## 2023-10-24 17:37:32.797809 INFO::Creating boxplot for categorical data, dysbiosisUC vs Ruminococcus.lactaris
## 2023-10-24 17:37:33.142767 INFO::Creating boxplot for categorical data, dysbiosisUC vs Burkholderiales.bacterium.1.1.47
## 2023-10-24 17:37:37.162861 INFO::Plotting data for metadata number 3, dysbiosisnonIBD
## 2023-10-24 17:37:37.165829 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Faecalibacterium.prausnitzii
## 2023-10-24 17:37:37.445241 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.rectale
## 2023-10-24 17:37:37.782888 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.sp.3.1.31
## 2023-10-24 17:37:38.154349 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.hominis
## 2023-10-24 17:37:38.492755 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Subdoligranulum.unclassified
## 2023-10-24 17:37:38.810953 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Lachnospiraceae.bacterium.3.1.46FAA
## 2023-10-24 17:37:39.136195 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Ruminococcus.torques
## 2023-10-24 17:37:39.475832 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Clostridium.leptum
## 2023-10-24 17:37:39.783935 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Odoribacter.splanchnicus
## 2023-10-24 17:37:40.073139 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Escherichia.coli
## 2023-10-24 17:37:40.448822 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Ruminococcus.obeum
## 2023-10-24 17:37:40.749199 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bilophila.unclassified
## 2023-10-24 17:37:41.087625 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.uniformis
## 2023-10-24 17:37:41.404718 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.eligens
## 2023-10-24 17:37:41.723766 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Klebsiella.pneumoniae
## 2023-10-24 17:37:42.050885 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Ruminococcus.bromii
## 2023-10-24 17:37:42.347327 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.vulgatus
## 2023-10-24 17:37:42.700151 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs C2likevirus.unclassified
## 2023-10-24 17:37:43.027531 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bifidobacterium.adolescentis
## 2023-10-24 17:37:43.340244 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Alistipes.finegoldii
## 2023-10-24 17:37:43.690635 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.inulinivorans
## 2023-10-24 17:37:44.020821 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Alistipes.onderdonkii
## 2023-10-24 17:37:44.338083 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.unclassified
## 2023-10-24 17:37:44.686124 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Eubacterium.hallii
## 2023-10-24 17:37:45.030765 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Roseburia.intestinalis
## 2023-10-24 17:37:45.375409 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Prevotella.copri
## 2023-10-24 17:37:45.739567 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.dorei
## 2023-10-24 17:37:46.049383 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Bacteroides.fragilis
## 2023-10-24 17:37:46.360642 INFO::Creating boxplot for categorical data, dysbiosisnonIBD vs Alistipes.shahii
## 2023-10-24 17:37:50.492882 INFO::Plotting data for metadata number 4, antibiotics
## 2023-10-24 17:37:50.494976 INFO::Creating boxplot for categorical data, antibiotics vs Roseburia.inulinivorans
## 2023-10-24 17:37:50.727334 INFO::Creating boxplot for categorical data, antibiotics vs Roseburia.hominis
## 2023-10-24 17:37:51.010524 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.rectale
## 2023-10-24 17:37:51.289363 INFO::Creating boxplot for categorical data, antibiotics vs Dialister.invisus
## 2023-10-24 17:37:51.546807 INFO::Creating boxplot for categorical data, antibiotics vs Roseburia.intestinalis
## 2023-10-24 17:37:51.912296 INFO::Creating boxplot for categorical data, antibiotics vs Dorea.longicatena
## 2023-10-24 17:37:52.213261 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.callidus
## 2023-10-24 17:37:52.494626 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.bromii
## 2023-10-24 17:37:52.808422 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.obeum
## 2023-10-24 17:37:53.085794 INFO::Creating boxplot for categorical data, antibiotics vs Klebsiella.pneumoniae
## 2023-10-24 17:37:53.41337 INFO::Creating boxplot for categorical data, antibiotics vs Bifidobacterium.adolescentis
## 2023-10-24 17:37:53.67311 INFO::Creating boxplot for categorical data, antibiotics vs Faecalibacterium.prausnitzii
## 2023-10-24 17:37:53.98091 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.hallii
## 2023-10-24 17:37:54.313659 INFO::Creating boxplot for categorical data, antibiotics vs Bilophila.unclassified
## 2023-10-24 17:37:54.592288 INFO::Creating boxplot for categorical data, antibiotics vs Clostridium.leptum
## 2023-10-24 17:37:54.884388 INFO::Creating boxplot for categorical data, antibiotics vs Lachnospiraceae.bacterium.3.1.46FAA
## 2023-10-24 17:37:55.150864 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.finegoldii
## 2023-10-24 17:37:55.459091 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.sp.3.1.31
## 2023-10-24 17:37:55.774251 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.fragilis
## 2023-10-24 17:37:56.005147 INFO::Creating boxplot for categorical data, antibiotics vs Alistipes.onderdonkii
## 2023-10-24 17:37:56.292325 INFO::Creating boxplot for categorical data, antibiotics vs Sutterella.wadsworthensis
## 2023-10-24 17:37:56.566083 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.eligens
## 2023-10-24 17:37:56.87823 INFO::Creating boxplot for categorical data, antibiotics vs Collinsella.aerofaciens
## 2023-10-24 17:37:57.191497 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.thetaiotaomicron
## 2023-10-24 17:37:57.473711 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroides.eggerthii
## 2023-10-24 17:37:57.802887 INFO::Creating boxplot for categorical data, antibiotics vs Haemophilus.parainfluenzae
## 2023-10-24 17:37:58.107393 INFO::Creating boxplot for categorical data, antibiotics vs Bifidobacterium.pseudocatenulatum
## 2023-10-24 17:37:58.373793 INFO::Creating boxplot for categorical data, antibiotics vs Ruminococcus.torques
## 2023-10-24 17:37:58.656525 INFO::Creating boxplot for categorical data, antibiotics vs Eubacterium.ventriosum
## 2023-10-24 17:37:58.948013 INFO::Creating boxplot for categorical data, antibiotics vs Parasutterella.excrementihominis
## 2023-10-24 17:37:59.190724 INFO::Creating boxplot for categorical data, antibiotics vs Peptostreptococcaceae.noname.unclassified
## 2023-10-24 17:37:59.511188 INFO::Creating boxplot for categorical data, antibiotics vs Veillonella.dispar
## 2023-10-24 17:37:59.827804 INFO::Creating boxplot for categorical data, antibiotics vs Veillonella.atypica
## 2023-10-24 17:38:00.118023 INFO::Creating boxplot for categorical data, antibiotics vs Bacteroidales.bacterium.ph8
## 2023-10-24 17:38:00.421841 INFO::Creating boxplot for categorical data, antibiotics vs Clostridium.nexile
## 2023-10-24 17:38:00.727822 INFO::Creating boxplot for categorical data, antibiotics vs Burkholderiales.bacterium.1.1.47
## 2023-10-24 17:38:01.010448 INFO::Creating boxplot for categorical data, antibiotics vs Lachnospiraceae.bacterium.1.1.57FAA
## 2023-10-24 17:38:01.335097 INFO::Creating boxplot for categorical data, antibiotics vs Akkermansia.muciniphila
## 2023-10-24 17:38:01.931057 INFO::Creating boxplot for categorical data, antibiotics vs Clostridium.citroniae
## 2023-10-24 17:38:02.196163 INFO::Creating boxplot for categorical data, antibiotics vs Odoribacter.splanchnicus
## 2023-10-24 17:38:05.444056 INFO::Plotting data for metadata number 5, age
## 2023-10-24 17:38:05.446758 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 17:38:05.761428 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 17:38:06.132588 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 17:38:06.422518 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 17:38:06.690487 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 17:38:06.966656 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 17:38:07.230493 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 17:38:07.555085 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 17:38:07.846307 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 17:38:08.114756 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 17:38:08.374945 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 17:38:08.64206 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 17:38:08.883033 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 17:38:09.167268 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 17:38:09.444263 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 17:38:09.741018 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 17:38:10.063491 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 17:38:10.370834 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 17:38:10.672997 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 17:38:10.964165 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 17:38:11.337243 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 17:38:11.645626 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 17:38:15.663491 INFO::Plotting data for metadata number 6, diagnosis
## 2023-10-24 17:38:15.666796 INFO::Creating boxplot for categorical data, diagnosis vs Bifidobacterium.adolescentis
## 2023-10-24 17:38:16.009547 INFO::Creating boxplot for categorical data, diagnosis vs Akkermansia.muciniphila
## 2023-10-24 17:38:16.360789 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.bolteae
## 2023-10-24 17:38:16.645415 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.putredinis
## 2023-10-24 17:38:17.023325 INFO::Creating boxplot for categorical data, diagnosis vs Coprobacillus.unclassified
## 2023-10-24 17:38:17.396507 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.clostridioforme
## 2023-10-24 17:38:17.741572 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.shahii
## 2023-10-24 17:38:18.111352 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.bromii
## 2023-10-24 17:38:18.468547 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.leptum
## 2023-10-24 17:38:18.773474 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.symbiosum
## 2023-10-24 17:38:19.086807 INFO::Creating boxplot for categorical data, diagnosis vs Roseburia.inulinivorans
## 2023-10-24 17:38:19.422901 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.bromii
## 2023-10-24 17:38:19.720726 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.shahii
## 2023-10-24 17:38:20.034974 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.finegoldii
## 2023-10-24 17:38:20.345987 INFO::Creating boxplot for categorical data, diagnosis vs Parabacteroides.goldsteinii
## 2023-10-24 17:38:20.638141 INFO::Creating boxplot for categorical data, diagnosis vs Bilophila.unclassified
## 2023-10-24 17:38:20.997995 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.leptum
## 2023-10-24 17:38:21.3958 INFO::Creating boxplot for categorical data, diagnosis vs Roseburia.hominis
## 2023-10-24 17:38:21.773867 INFO::Creating boxplot for categorical data, diagnosis vs Coprobacillus.unclassified
## 2023-10-24 17:38:22.118035 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.onderdonkii
## 2023-10-24 17:38:22.458175 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.ventriosum
## 2023-10-24 17:38:22.766315 INFO::Creating boxplot for categorical data, diagnosis vs Roseburia.hominis
## 2023-10-24 17:38:23.063772 INFO::Creating boxplot for categorical data, diagnosis vs Sutterella.wadsworthensis
## 2023-10-24 17:38:23.363953 INFO::Creating boxplot for categorical data, diagnosis vs Akkermansia.muciniphila
## 2023-10-24 17:38:23.624144 INFO::Creating boxplot for categorical data, diagnosis vs Sutterella.wadsworthensis
## 2023-10-24 17:38:23.972538 INFO::Creating boxplot for categorical data, diagnosis vs Subdoligranulum.unclassified
## 2023-10-24 17:38:24.355771 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.symbiosum
## 2023-10-24 17:38:24.647367 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.onderdonkii
## 2023-10-24 17:38:24.934622 INFO::Creating boxplot for categorical data, diagnosis vs Parabacteroides.unclassified
## 2023-10-24 17:38:25.24312 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.lactaris
## 2023-10-24 17:38:25.50706 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.callidus
## 2023-10-24 17:38:25.809355 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.gnavus
## 2023-10-24 17:38:26.114603 INFO::Creating boxplot for categorical data, diagnosis vs Bacteroides.fragilis
## 2023-10-24 17:38:26.412292 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.rectale
## 2023-10-24 17:38:26.702093 INFO::Creating boxplot for categorical data, diagnosis vs Odoribacter.splanchnicus
## 2023-10-24 17:38:26.969458 INFO::Creating boxplot for categorical data, diagnosis vs Parabacteroides.distasonis
## 2023-10-24 17:38:27.248886 INFO::Creating boxplot for categorical data, diagnosis vs Eubacterium.rectale
## 2023-10-24 17:38:27.535238 INFO::Creating boxplot for categorical data, diagnosis vs Bacteroides.finegoldii
## 2023-10-24 17:38:27.797024 INFO::Creating boxplot for categorical data, diagnosis vs Ruminococcus.callidus
## 2023-10-24 17:38:28.044963 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.finegoldii
## 2023-10-24 17:38:28.323462 INFO::Creating boxplot for categorical data, diagnosis vs Alistipes.putredinis
## 2023-10-24 17:38:28.595128 INFO::Creating boxplot for categorical data, diagnosis vs Clostridium.bolteae
## 2023-10-24 17:38:28.855185 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 Patched (2023-06-17 r84564)
## Platform: x86_64-apple-darwin20 (64-bit)
## Running under: macOS Monterey 12.6.5
## 
## Matrix products: default
## BLAS:   /Library/Frameworks/R.framework/Versions/4.3-x86_64/Resources/lib/libRblas.0.dylib 
## LAPACK: /Library/Frameworks/R.framework/Versions/4.3-x86_64/Resources/lib/libRlapack.dylib;  LAPACK version 3.11.0
## 
## locale:
## [1] C/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
## 
## time zone: America/New_York
## tzcode source: internal
## 
## attached base packages:
## [1] stats     graphics  grDevices utils     datasets  methods   base     
## 
## other attached packages:
## [1] Maaslin2_1.16.0
## 
## 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.