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Title:Metrological evaluation of DNA extraction method effects on the bacterial microbiome and resistome in sputum
Authors:ID Benčič, Aleksander (Author)
ID Toplak, Nataša (Author)
ID Koren, Simon (Author)
ID Bogožalec Košir, Alexandra (Author)
ID Milavec, Mojca (Author)
ID Tomič, Viktorija (Author)
ID Lužnik, Dane (Author)
ID Dreo, Tanja (Author)
Files:URL URL - Source URL, visit https://doi.org/10.1128/msystems.00735-24
 
.pdf PDF - Presentation file, download (996,30 KB)
MD5: 1514D5B89BB40CF0A116D4A0F6E8A28B
 
Language:English
Typology:1.01 - Original Scientific Article
Organization:Logo NIB - National Institute of Biology
Abstract:Targeted high-throughput sequencing (HTS) has revolutionized the way we look at bacterial communities. It can be used for the species-specific detection of bacteria as well as for the determination of the microbiome and resistome and can be applied to samples from almost any environment. However, the results of targeted HTS can be influenced by many factors, which poses a major challenge for its use in clinical diagnostics. In this study, we investigated the impact of the DNA extraction method on the determination of the bacterial microbiome and resistome by targeted HTS using principles from metrology and diagnostics such as repeatability and analytical sensitivity. Sputum samples spiked with Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa at three different concentrations (103–106 cells/mL) were used. DNA was extracted from each sample on 2 separate days in three replicates each using three different extraction methods based on cetrimonium bromide, magnetic beads, and silica membranes. All three spiked bacteria were detected in sputum, and the DNA extraction method had no significant effect on detection. However, the DNA extraction method had significant effects on the composition of the microbiome and the resistome. The sequencing results were repeatable in the majority of cases. The silica membrane-based DNA extraction kit provided the most repeatable results and the highest diversity of the microbiome and resistome. Targeted HTS has been shown to be a reliable tool for determining the microbiome and resistome; however, the method of DNA extraction should be carefully selected to minimize its impact on the results.
Keywords:targeted high-throughput sequencing, bacterial microbiome, resistome, bacteria detection, DNA extraction, metrology, diagnostics, repeatability
Publication status:Published
Publication version:Version of Record
Publication date:16.08.2024
Year of publishing:2024
Number of pages:str. 1-16
PID:20.500.12556/DiRROS-20255 New window
UDC:579.6
ISSN on article:2379-5077
DOI:10.1128/msystems.00735-24 New window
COBISS.SI-ID:205913091 New window
Note:Nasl. z nasl. zaslona; Soavtorji: Nataša Toplak, Simon Koren, Alexandra Bogožalec Košir, Mojca Milavec, Viktorija Tomič, Dane Lužnik, Tanja Dreo; Članek v PDF formatu obsega 16 str.; Online first: 16 Avg. 2024; Opis vira z dne 31. 8. 2024;
Publication date in DiRROS:30.08.2024
Views:494
Downloads:341
Metadata:XML DC-XML DC-RDF
:
BENČIČ, Aleksander, TOPLAK, Nataša, KOREN, Simon, BOGOŽALEC KOŠIR, Alexandra, MILAVEC, Mojca, TOMIČ, Viktorija, LUŽNIK, Dane and DREO, Tanja, 2024, Metrological evaluation of DNA extraction method effects on the bacterial microbiome and resistome in sputum. mSystems [online]. 2024. P. 1–16. [Accessed 6 April 2025]. DOI 10.1128/msystems.00735-24. Retrieved from: https://dirros.openscience.si/IzpisGradiva.php?lang=eng&id=20255
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Record is a part of a journal

Title:mSystems
Shortened title:mSystems
Publisher:American Society for Microbiology, 2016-
ISSN:2379-5077
COBISS.SI-ID:525849369 New window

Document is financed by a project

Funder:ARIS - Slovenian Research and Innovation Agency
Project number:P4-0165-2022
Name:Biotehnologija in sistemska biologija rastlin
Funder:ARIS - Slovenian Research and Innovation Agency
Project number:Z2-1860-2019
Name:Meroslovje za sledenje bolnišničnih okužb respiratornega trakta, ki jih povzročajo Gram-negativne bakterije
Funder:EC - European Commission
Funder:ARIS - Slovenian Research and Innovation Agency
Project number:52760
Funder:EC - European Commission
Funding programme:EMPIR program co-financed by the participating states and from the European Union’s Horizon 2020 research and innovation program
Project number:18HLT03
Acronym:SEPTIMET
Funder:Other - Other funder or multiple funders
Funding programme:Metrology Institute of the Republic of Slovenia (MIRS), with financial support from the European Regional Development Fund

Licences

License:CC BY 4.0, Creative Commons Attribution 4.0 International
Link:http://creativecommons.org/licenses/by/4.0/
Description:This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

Language:Slovenian
Keywords:tarčno visokozmogljivo sekvenciranje, bakterijski mikrobiom, rezistom, detekcija bakterij, izolacija DNA, meroslovje, diagnostika, ponovljivost


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