B-GUT reference genome database improves biomarker discovery and fungal identification in gut metagenomes

Background Accurate taxonomy assignment to sequencing reads is a key step in metagenomic studies, impacting all downstream analyses. The accuracy of this step critically depends on the quality and comprehensiveness of the used reference genome database. While fungi are ubiquitous and relevant in the human microbiome, they are generally poorly represented in current databases. To address this and other limitations, we developed B-GUT, a custom Kraken2 database that integrates i) a broad and curated collection of 2,110 fungal reference genomes; ii) the human telomere-to-telomere reference genome; and iii) two available curated databases for gut-specific bacterial and archaeal genomes. Results Our analysis of publicly available fungal genomes revealed significant contamination and substantial cross-mapping of human sequencing reads to fungal genome references, underscoring the necessity of rigorous curation and accurate host read filtering. We validated our genome curation pipeline and the resulting B-GUT database using mock microbial communities with known compositions. Finally, we showcased the utility of B-GUT by re-analysing data from a published colorectal cancer metagenomics study, where its use led to significantly improved results, providing more precise taxonomic assignments and a more accurate identification of differentially abundant taxa for both bacterial and fungal communities. Conclusions We introduce B-GUT, a reference genome database centered on the gut microbiome, featuring a uniquely curated and comprehensive collection of fungal genomes, often underrepresented in existing resources. We demonstrate the importance of database curation and the enhanced capacity of B-GUT for identifying biologically relevant microbes.