This repository contains the code to reproduce the analysis in the study investigating the effects of gut microbiota on Bacillus Calmette-Guerin (BCG) vaccination of 321 healthy Dutch individuals. The results are presented in the paper The influence of the gut microbiome on BCG-induced trained immunity by Martin Stražar, Vera P. Mourits, Valerie A.C.M. Koeken, L. Charlotte J. de Bree, Simone J.C.F.M. Moorlag, Leo A.B. Joosten, Reinout van Crevel, Hera Vlamakis, Mihai G. Netea, Ramnik J. Xavier (2021) The bacillus Calmette-Guérin (BCG) vaccine protects against tuberculosis and heterologous infections but elicits high interindividual variation in specific and nonspecific (trained) immune responses. While the gut microbiome is increasingly recognized as an important modulator of vaccine responses and immunity in general, its potential role in BCG-induced protection is largely unknown. Stool and blood were collected from 321 healthy adults before BCG vaccination, followed by blood sampling two weeks and three months afterwards. Metagenomics based on de novo genome assembly revealed 43 immunomodulatory taxa. The nonspecific, trained immune response was detected by altered production of cytokines IL-6, IL-1β, and TNF-α upon ex vivo blood restimulation with Staphylococcus aureus and negatively correlated with abundance of Roseburia. The specific response, measured by IFN-γ production upon Mycobacterium tuberculosis stimulation, was associated positively with Ruminococcus and Eggerthella lenta. The immunomodulatory taxa identified also had the strongest effects on circulating metabolites, with Roseburia predominantly affecting phenylalanine metabolism. This was corroborated by abundances of relevant enzymes, suggesting alternate phenylalanine metabolism modules are activated in a Roseburia species-dependent manner. Variability in cytokine production after BCG vaccination was associated with the abundance of microbial genomes, which in turn affect or produce metabolites in circulation. Roseburia was found to alter both trained immune responses and phenylalanine metabolism, revealing microbes and microbial products that may alter BCG-induced immunity. Together, our findings contribute to the understanding of specific and trained immune responses after BCG vaccination. The analysis and dataset details are further described in README.md and https://gitlab.com/xavier-lab-computation/public/bcg300 .