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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\u2010whole blood (WB) and plasma samples, using contrived blood specimens spiked with bacteria for shotgun metagenomics diagnostics via Oxford Nanopore sequencing and PCR\u2010based library preparation. Results showed that bacterial reads were higher in WB than plasma. Differences for <jats:styled-content style=\"fixed-case\"><jats:italic>Staphylococcus aureus<\/jats:italic><\/jats:styled-content> and <jats:styled-content style=\"fixed-case\"><jats:italic>Streptococcus pneumoniae<\/jats:italic><\/jats:styled-content> were more pronounced compared to <jats:styled-content style=\"fixed-case\"><jats:italic>Escherichia coli<\/jats:italic><\/jats:styled-content>. 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\u2010positive bacteria than Gram\u2010negative, suggesting that the human DNA depletion exerts a negative effect on Gram\u2010negative 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\u2010based diagnosis of blood stream infections using Nanopore sequencing.<\/jats:p>","DOI":"10.1111\/apm.13511","type":"journal-article","created":{"date-parts":[[2025,1,14]],"date-time":"2025-01-14T03:36:05Z","timestamp":1736825765000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Critical Steps in Shotgun Metagenomics\u2010Based Diagnosis of Bloodstream Infections Using Nanopore Sequencing"],"prefix":"10.1111","volume":"133","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-6450-647X","authenticated-orcid":false,"given":"Amelia","family":"Bj\u00f6rnberg","sequence":"first","affiliation":[{"name":"Department of Laboratory Medicine Clinical Microbiology \u00d6rebro University Hospital and Faculty of Medicine and Health at \u00d6rebro University  \u00d6rebro 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