Enriching the Human Stool Microeukaryotes for Shotgun Sequencing

The human gut microbiome harbours a diverse community of microeukaryotes, predominantly fungi, which may potentially play important roles in gut ecology and homeostasis. Despite their potential, the study of gut microeukaryotes has been hampered by the limited sensitivity of standard sequencing approaches, which struggle to capture DNA from low-abundance microorganisms against the overwhelming background of bacterial biomass. To address this, we developed a method to selectively enrich for microeukaryotic cells in human faecal samples by depleting bacterial cells prior to metagenomic sequencing. Through systematic comparison and optimisation at each processing step, we established a robust standard operating procedure (SOP) for microeukaryotic cell enrichment. By benchmarking this SOP across eight human faecal samples with three technical replicates each, we showed that it consistently increased microeukaryote representation in metagenomic libraries, greater microeukaryotic taxonomic diversity, and a reduced proportion of unclassified taxa. Together, these improvements enabled substantially deeper characterisation of the microeukaryotic fraction of the human gut microbiome.