Metagenomic pathway differential activity analysis and graphical representation (picrust2_plot.R)

Finally, after inferring on metagenomic composition with PICRUSt2, this script performs an ANOVA-like differential expression analysis through ALDEx2 tool with Benjamini-Hochberg (BH) p-values adjustment. Also, this tool generates two graphical visualizations through ggpicrust2 package:

• An errorbar visualization with significant metagenomic pathways between groups
• A PCA visualization of the metagenomic pathways composition comparing groups

As well as we did in the other R scripts, you must deactivate conda picrust2 enviroment prior to make this analysis in R:

conda deactivate

You can run this script as shown below:

Rscript /file_path/picrust2_plot.R \
--metadata_fp /file_path/metadata.tsv \
--col Comparison \
--ref_group Healthy \
--method MetaCyc \
--picrust_dir /directory_path/picrust2/output \
--Visualizations /directory_path/picrust2/Visualizations

Specifying the variables:

• --metadata_fp - Metadata file path
• --col - A character string indicating the metadata column where the groups to compare are
• --ref_group - Reference group for statistical analysis
• --method - Selected PICRUSt2 inference method for statistical analysis. These methods can be: KO for KEGG Orthologs, EC for Enzyme Commission Numbers and MetaCyc for MetaCyc pathways inference.
• --picrust_dir - PICRUSt2 output directory for detecting input files. It is generated automatically with picrust2.sh script
• --Visualizations - PICRUSt2 "Visualizations" directory for output files generation. It is generated automatically with picrust2.sh script

This script returns:

• method.tsv - A data frame with the results of pathway differential abundance analysis with p-values corrected by BH method. This file is called KO, EC or MetaCyc depending on the selected method.
• method.png - A graphical errorbar visualization with the significant features (only if there are significant pathways).
• method_PCA.png A graphical PCA visualization comparing metagenomic pathway composition of groups