Development and optimization of the host DNA depletion in blood cultures using a saponin and SAN nucleases-based method

Bloodstream infections and sepsis are major health issues causing millions of deaths annually. Early and accurate diagnosis of sepsis is crucial for reducing mortality and combating antimicrobial resistance. However, current diagnostic methods are slow and time-consuming. Nanopore sequencing has the potential to serve as a rapid diagnostic method for sepsis. However, extracting bacterial DNA from blood samples is challenging due to the high content of host DNA. This study aimed to develop and optimize a method using saponin and SAN nucleases for effective host DNA depletion in blood cultures. Different concentrations of saponin, HL-SAN, and M-SAN nucleases, combined with various salt conditions (NaCl and MgCl2), were tested for their ability to deplete host DNA in blood cultures spiked with E. coli and S. aureus. The impact of different bead beating durations on DNA fragment sizes was also examined. The efficiency of host DNA depletion and bacterial DNA extraction was assessed using quantitative PCR (qPCR) and nanopore sequencing. Results indicated that 4% of saponin effectively lysed host cells, releasing DNA while preserving bacterial cells. The host DNA is then efficiently depleted using 250 units (10 L) of HL-SAN and M-SAN nucleases. Although SAN in combination with higher salt concentrations (2.5 M NaCl and 50 mM MgCl2) showed somewhat better host DNA removal, the difference was not statistically significant. Reducing bead beating time to 6 minutes improved the recovery of longer DNA fragments compared to 10 minutes. These results demonstrate an effective method for host DNA depletion using saponin and SAN nucleases, which is compatible with downstream processes, such as nanopore sequencing and qPCR.