DSN-based host DNA depletion for simultaneous detection of DNA and RNA pathogens in next-generation sequencing

In recent years, nucleic acid sequencing has been increasingly used in clinical pathogen detection. Typically, 20-100 million metagenomic reads are generated per sample, but 90%-99% originate from host nucleic acids, leaving only 1-2 million for microbial identification-potentially compromising detection sensitivity. Here, we present a novel duplex-specific nuclease (DSN)-based method to reduce host nucleic acid content in next-generation sequencing. Validated using mock communities and clinical samples, it yielded a 2- to 3-fold increase in pathogen RPM (reads per million) in metagenomic NGS (mNGS) and a more pronounced 3- to 10-fold improvement in probe-based targeted NGS (tNGS). Optimal performance was achieved with: 65°C annealing temperature; 2 μg Cot-1 DNA and 50 μmol rProbe as driver DNA; and 150 mmol·L-1 tetramethylammonium chloride (TMAC)-using 25 ng input nucleic acid for library preparation. The DSN workflow adds less than 30 min to library preparation and enables rapid, low-loss microbial enrichment, enhancing pathogen detection in clinical NGS. While effective, its host depletion efficiency could be further improved by expanding driver DNA coverage to additional abundant human genomic regions, such as LINE and LTR repeats.