Nucleosome-targeted host DNA depletion enables automated plasma metagenomic sequencing for sensitive detection of bloodstream pathogens

Bloodstream infections (BSIs) are leading causes of sepsis-related mortality. Although metagenomic next-generation sequencing (mNGS) enables culture-independent pathogen detection, its clinical utility in plasma is limited by the overwhelming abundance of host cell-free DNA (cfDNA) and labor-intensive manual workflows. A plasma host DNA depletion mNGS (HD-mNGS) assay was developed which integrated nucleosome-targeted host DNA depletion with automated DNA extraction and library preparation. Analytical performance was evaluated through limit of detection, linearity, precision, and contamination control. Clinical performance was assessed in a cohort of 107 patients with suspected BSI and benchmarked against blood culture (BC), conventional microbiological testing (CMT), and standard mNGS without host depletion, using a composite clinical reference standard. Nucleosome depletion markedly reduced host DNA background by an average of 66-fold, consequently enriching microbial reads by approximately 46.73-fold. The automated HD-mNGS assay exhibited robust analytical sensitivity, with limits of detection (LoD) ranging from 9.1 to 38 genome equivalents (GE) /mL for bacteria and fungi, and from 283 to 321 GE/mL for viruses and excellent linearity across tested concentrations (R 2 = 0.915–0.989). Furthermore, the automated workflow maintained strong quantitative correlation with manual protocols while significantly reducing common skin and environmental contaminants by 71.7% and 83.7%, respectively. In a cohort of 107 patients, HD-mNGS demonstrates improved diagnostic performance for BSI, achieving a significantly higher pathogen detection rate (64.49%) and clinical positive percent agreement (PPA: 95.24%) than standard mNGS, BC, and CMT ( P < 0.001 ). Crucially, HD-mNGS demonstrates enhanced performance in detecting rare, fastidious, and intracellular pathogens (such as Mycobacterium tuberculosis and Rickettsia ) that yield extremely low concentrations of circulating DNA, overcoming the limitations of traditional methods while maintaining high overall diagnostic total percent agreement (TPA: 88.79%). Nucleosome-targeted host DNA depletion integrated with a fully automated mNGS platform significantly enhances microbial detection in plasma and provides a scalable approach for standardized BSI diagnostics.