Enhanced Computational and Experimental Approaches for Comparative Analysis of the Human Mycobiome

The mycobiome is now recognized as a critical part of the human microbial ecosystem, playing a significant role in both health and disease. Mycobiome research is currently in its infancy and relies on fungal-specific primers with challenges in bioinformatics to accurately determine the structure of the mycobiota community. In addition, the majority of computational and experimental methods currently in use have been optimized for bacteria rather than for fungi. Here, we provide a comparative analysis of extraction methodologies for metagenome sequencing and subsequent bioinformatic analysis that will enhance fungal species identification. We utilized cultured mock fungal communities, including both yeast and mold species, to evaluate the efficiency of extraction protocols. We further enhanced computational analysis of the mycobiome, using mock and human metagenome data together with our curated catalogue consisting of 984 fungal genomes specific to the human mycobiome. Application of this optimised workflow for oral and gut samples of healthy individuals detected of novel species from Puccinia and Lentinus genera among the established presence of Malassezia , Rhizophagus , Candida and Saccharomyces genera. In addition, Enterocytozoon, was identified specifically in the gut mycobiome. Our pipeline enabled the comprehensive sampling of all fungal genes in a microbial community within a human sample minimising bias, reducing errors and artefacts from amplification, and providing accurate diversity and abundance data for the human mycobiome.