Amelia Björnberg, David Nestor, Nilay Peker, Bhanu Sinha, Natacha Couto, John Rossen, Martin Sundqvist, Paula Mölling
{"title":"使用纳米孔测序的基于散弹枪宏基因组学的血流感染诊断的关键步骤。","authors":"Amelia Björnberg, David Nestor, Nilay Peker, Bhanu Sinha, Natacha Couto, John Rossen, Martin Sundqvist, Paula Mölling","doi":"10.1111/apm.13511","DOIUrl":null,"url":null,"abstract":"<p>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 <i>Staphylococcus aureus</i> and <i>Streptococcus pneumoniae</i> were more pronounced compared to <i>Escherichia coli</i>. 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.</p>","PeriodicalId":8167,"journal":{"name":"Apmis","volume":"133 1","pages":""},"PeriodicalIF":2.2000,"publicationDate":"2025-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11730497/pdf/","citationCount":"0","resultStr":"{\"title\":\"Critical Steps in Shotgun Metagenomics-Based Diagnosis of Bloodstream Infections Using Nanopore Sequencing\",\"authors\":\"Amelia Björnberg, David Nestor, Nilay Peker, Bhanu Sinha, Natacha Couto, John Rossen, Martin Sundqvist, Paula Mölling\",\"doi\":\"10.1111/apm.13511\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>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 <i>Staphylococcus aureus</i> and <i>Streptococcus pneumoniae</i> were more pronounced compared to <i>Escherichia coli</i>. 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.</p>\",\"PeriodicalId\":8167,\"journal\":{\"name\":\"Apmis\",\"volume\":\"133 1\",\"pages\":\"\"},\"PeriodicalIF\":2.2000,\"publicationDate\":\"2025-01-14\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11730497/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Apmis\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1111/apm.13511\",\"RegionNum\":4,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"IMMUNOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Apmis","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1111/apm.13511","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"IMMUNOLOGY","Score":null,"Total":0}
Critical Steps in Shotgun Metagenomics-Based Diagnosis of Bloodstream Infections Using Nanopore Sequencing
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.
期刊介绍:
APMIS, formerly Acta Pathologica, Microbiologica et Immunologica Scandinavica, has been published since 1924 by the Scandinavian Societies for Medical Microbiology and Pathology as a non-profit-making scientific journal.