Critical Steps in Shotgun Metagenomics-Based Diagnosis of Bloodstream Infections Using Nanopore Sequencing.
Amelia Bj��rnberg, David Nestor, Nilay Peker, Bhanu Sinha, Natacha Couto, John Rossen, Martin Sundqvist, Paula M��lling
Author Information
Amelia Bj��rnberg: Department of Laboratory Medicine, Clinical Microbiology ��rebro University Hospital and Faculty of Medicine and Health at ��rebro University, ��rebro, Sweden. ORCID
David Nestor: Department of Laboratory Medicine, Clinical Microbiology ��rebro University Hospital and Faculty of Medicine and Health at ��rebro University, ��rebro, Sweden.
Nilay Peker: Department of Medical Microbiology and Infection Prevention, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands.
Bhanu Sinha: Department of Medical Microbiology and Infection Prevention, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands.
Natacha Couto: Department of Medical Microbiology and Infection Prevention, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands.
John Rossen: Department of Medical Microbiology and Infection Prevention, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands. ORCID
Martin Sundqvist: Department of Laboratory Medicine, Clinical Microbiology ��rebro University Hospital and Faculty of Medicine and Health at ��rebro University, ��rebro, Sweden.
Paula M��lling: Department of Laboratory Medicine, Clinical Microbiology ��rebro University Hospital and Faculty of Medicine and Health at ��rebro University, ��rebro, Sweden.
Shotgun metagenomics offers a broad detection of pathogens for rapid blood stream infection of pathogens but struggles with often low numbers of pathogens combined with high levels of human background DNA in clinical samples. This study aimed to develop a shotgun metagenomics protocol using blood spiked with various bacteria and to assess bacterial DNA extraction efficiency with human DNA depletion. The Blood Pathogen Kit (Molzym) was used to extract DNA from EDTA-whole blood (WB) and plasma samples, using contrived blood specimens spiked with bacteria for shotgun metagenomics diagnostics via Oxford Nanopore sequencing and PCR-based library preparation. Results showed that bacterial reads were higher in WB than plasma. Differences for Staphylococcus aureus and Streptococcus pneumoniae were more pronounced compared to Escherichia coli. Plasma samples exhibited better method reproducibility, with more consistent droplet digital PCR results for human DNA. The study found that extraction was more efficient for Gram-positive bacteria than Gram-negative, suggesting that the human DNA depletion exerts a negative effect on Gram-negative bacteria. Overall, shotgun metagenomics needs further optimisation to improve bacterial DNA recovery and enhance pathogen detection sensitivity. This study highlights some critical steps in the methodology of shotgun metagenomic-based diagnosis of blood stream infections using Nanopore sequencing.
References
Int J Mol Sci. 2022 Feb 16;23(4):
[PMID: 35216302]
