Abstract
<jats:p>Metagenomic whole genome sequencing is a powerful culture-independent approach for pathogen detection and microbiome analysis. However, sensitivity and cost-effectiveness are often limited by high levels of host DNA in the sequenced samples. Here, we describe a novel bead-based host-depletion method, adapted from a respiratory metagenomics workflow, in which selective physical disruption of host cells is followed by magnetic bead capture of released host DNA, while intact bacterial cells are retained in the supernatant for downstream extraction. We evaluated this bead-based method using mock samples, a bacterial dilution series, and clinical specimens. In mock samples with Staphylococcus aureus, Escherichia coli or Pseudomonas aeruginosa combined with THP-1 human cells, bead-based depletion host DNA by up to 99% and significantly improved the ratio of bacterial-to-host DNA, although bacterial DNA was also significantly reduced. A serial dilution experiment showed that performance was strongly influenced by bacterial biomass, with the greatest benefit observed when bacterial input was moderate-to-high. In clinical samples, host-derived reads were reduced across all samples; in some cases, this enabled the detection of pathogens above the diagnostic abundance threshold. Our findings demonstrate that bead-based host depletion is a promising approach for enhancing microbial representation in metagenomic samples.</jats:p>