| Title: | Metrological evaluation of DNA extraction method effects on the bacterial microbiome and resistome in sputum |
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| 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) |
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| Files: |  URL - Source URL, visit https://doi.org/10.1128/msystems.00735-24
 PDF - Presentation file, download (996,19 KB)
MD5: 83231A83E599A39435FD6FEFE212C6AA
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| Language: | English |
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| Typology: | 1.01 - Original Scientific Article |
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| Organization: |  NIB - National Institute of Biology
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| 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. |
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| Keywords: | targeted high-throughput sequencing, bacterial microbiome, resistome, bacteria detection, DNA extraction, metrology, diagnostics, repeatability |
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| Publication status: | In print |
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| Publication version: | Version of Record |
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| Publication date: | 16.08.2024 |
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| Year of publishing: | 2024 |
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| Number of pages: | str. 1-16 |
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| PID: | 20.500.12556/DiRROS-20255  |
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| UDC: | 579.6 |
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| ISSN on article: | 2379-5077 |
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| DOI: | 10.1128/msystems.00735-24 |
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| COBISS.SI-ID: | 205913091 |
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| 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;
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| Publication date in DiRROS: | 30.08.2024 |
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| Views: | 264 |
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| Downloads: | 171 |
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