# R script for “A low FODMAP diet is associated with changes in the microbiota and reduction in 
# breath hydrogen but not colonic volume in healthy subjects”

# The majority of the results were produced with the R package "mare" (https://github.com/katrikorpela/mare). 
# The functions that were used for statistical analysis include "GroupTest" and "CovariateTest" 
# Examples of the function usage is provided below.

# This source code does not include finalization of figures, font settings etc..



# GroupTest, finds differences in bacterial abundance between selected groups

GroupTest(taxonomic.table = ".../taxonomic.table.txt", #provide bacterial taxonomic data
                       meta = ".../meta.file.txt", 
                       group = "group", # grouping variable
                       compare.to = "BL", 
                       readcount.cutoff = 5000, #sample with reads below 10000 were excluded
                       confounders = c('Age', 'BMI'), 
                       subject.ID = 'SubjectID', 
                       outlier.cutoff = 3, # values with SD over 3 were given the cut-off values to eliminate disproportional effects of outliers
                       p.cutoff = 0.05 #taxa with p-value below 0.05 plotted
                       )


# Covariate test, estimates the significance of the association between each bacterial taxon and a continuous covariate.

CovariateTest(taxonomic.table = ".../taxonomic.table.txt", #provide bacterial taxonomic data
                       meta = ".../meta.file.txt", 
                       covariate="TotalVolume", #Covariate of interest
                       readcount.cutoff = 5000, #sample with reads below 5000 were excluded
                       confounders = c('Age', 'BMI'), 
                       subject.ID = 'SubjectID', 
                       outlier.cutoff = 3, # values with SD over 3 were given the cut-off values to eliminate disproportional effects of outliers
                       p.cutoff = 0.05 #taxa with p-value below 0.05 plotted
                       )

# More information and more commands at https://github.com/katrikorpela/mare