Pragmatic Considerations When Extracting DNA for Metagenomics Analyses of Clinical Samples
Microbiome analyses are essential for understanding microorganism composition and diversity, but interpretation is often challenging due to biological and technical variables. DNA extraction is a critical step that can significantly bias results, particularly in samples containing a high abundance o...
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MDPI AG
2023-07-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/14/11262 |
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| author | Claudio Neidhöfer Maria Bagniceva Nina Wetzig Martin A. Sieber Ralf Thiele Marijo Parčina |
| author_facet | Claudio Neidhöfer Maria Bagniceva Nina Wetzig Martin A. Sieber Ralf Thiele Marijo Parčina |
| author_sort | Claudio Neidhöfer |
| collection | DOAJ |
| description | Microbiome analyses are essential for understanding microorganism composition and diversity, but interpretation is often challenging due to biological and technical variables. DNA extraction is a critical step that can significantly bias results, particularly in samples containing a high abundance of challenging-to-lyse microorganisms. Taking into consideration the distinctive microenvironments observed in different bodily locations, our study sought to assess the extent of bias introduced by suboptimal bead-beating during DNA extraction across diverse clinical sample types. The question was whether complex targeted extraction methods are always necessary for reliable taxonomic abundance estimation through amplicon sequencing or if simpler alternatives are effective for some sample types. Hence, for four different clinical sample types (stool, cervical swab, skin swab, and hospital surface swab samples), we compared the results achieved from extracting targeted manual protocols routinely used in our research lab for each sample type with automated protocols specifically not designed for that purpose. Unsurprisingly, we found that for the stool samples, manual extraction protocols with vigorous bead-beating were necessary in order to avoid erroneous taxa proportions on all investigated taxonomic levels and, in particular, false under- or overrepresentation of important genera such as <i>Blautia, Faecalibacterium,</i> and <i>Parabacteroides.</i> However, interestingly, we found that the skin and cervical swab samples had similar results with all tested protocols. Our results suggest that the level of practical automation largely depends on the expected microenvironment, with skin and cervical swabs being much easier to process than stool samples. Prudent consideration is necessary when extending the conclusions of this study to applications beyond rough estimations of taxonomic abundance. |
| first_indexed | 2024-03-11T01:02:09Z |
| format | Article |
| id | doaj.art-e35fa10e62fd497f82a0c28306ba4437 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T01:02:09Z |
| publishDate | 2023-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-e35fa10e62fd497f82a0c28306ba44372023-11-18T19:36:34ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-07-0124141126210.3390/ijms241411262Pragmatic Considerations When Extracting DNA for Metagenomics Analyses of Clinical SamplesClaudio Neidhöfer0Maria Bagniceva1Nina Wetzig2Martin A. Sieber3Ralf Thiele4Marijo Parčina5Institute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, Venusberg Campus 1, 53127 Bonn, GermanyInstitute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, Venusberg Campus 1, 53127 Bonn, GermanyInstitute for Functional Gene Analytics, Bonn-Rhein-Sieg University of Applied Sciences, 53757 Sankt Augustin, GermanyInstitute for Functional Gene Analytics, Bonn-Rhein-Sieg University of Applied Sciences, 53757 Sankt Augustin, GermanyInstitute for Functional Gene Analytics, Bonn-Rhein-Sieg University of Applied Sciences, 53757 Sankt Augustin, GermanyInstitute of Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, Venusberg Campus 1, 53127 Bonn, GermanyMicrobiome analyses are essential for understanding microorganism composition and diversity, but interpretation is often challenging due to biological and technical variables. DNA extraction is a critical step that can significantly bias results, particularly in samples containing a high abundance of challenging-to-lyse microorganisms. Taking into consideration the distinctive microenvironments observed in different bodily locations, our study sought to assess the extent of bias introduced by suboptimal bead-beating during DNA extraction across diverse clinical sample types. The question was whether complex targeted extraction methods are always necessary for reliable taxonomic abundance estimation through amplicon sequencing or if simpler alternatives are effective for some sample types. Hence, for four different clinical sample types (stool, cervical swab, skin swab, and hospital surface swab samples), we compared the results achieved from extracting targeted manual protocols routinely used in our research lab for each sample type with automated protocols specifically not designed for that purpose. Unsurprisingly, we found that for the stool samples, manual extraction protocols with vigorous bead-beating were necessary in order to avoid erroneous taxa proportions on all investigated taxonomic levels and, in particular, false under- or overrepresentation of important genera such as <i>Blautia, Faecalibacterium,</i> and <i>Parabacteroides.</i> However, interestingly, we found that the skin and cervical swab samples had similar results with all tested protocols. Our results suggest that the level of practical automation largely depends on the expected microenvironment, with skin and cervical swabs being much easier to process than stool samples. Prudent consideration is necessary when extending the conclusions of this study to applications beyond rough estimations of taxonomic abundance.https://www.mdpi.com/1422-0067/24/14/11262human microbiomemicrobiome analysesmicrobial community structureDNA extraction protocolsextraction-linked biasautomation of sample processing |
| spellingShingle | Claudio Neidhöfer Maria Bagniceva Nina Wetzig Martin A. Sieber Ralf Thiele Marijo Parčina Pragmatic Considerations When Extracting DNA for Metagenomics Analyses of Clinical Samples International Journal of Molecular Sciences human microbiome microbiome analyses microbial community structure DNA extraction protocols extraction-linked bias automation of sample processing |
| title | Pragmatic Considerations When Extracting DNA for Metagenomics Analyses of Clinical Samples |
| title_full | Pragmatic Considerations When Extracting DNA for Metagenomics Analyses of Clinical Samples |
| title_fullStr | Pragmatic Considerations When Extracting DNA for Metagenomics Analyses of Clinical Samples |
| title_full_unstemmed | Pragmatic Considerations When Extracting DNA for Metagenomics Analyses of Clinical Samples |
| title_short | Pragmatic Considerations When Extracting DNA for Metagenomics Analyses of Clinical Samples |
| title_sort | pragmatic considerations when extracting dna for metagenomics analyses of clinical samples |
| topic | human microbiome microbiome analyses microbial community structure DNA extraction protocols extraction-linked bias automation of sample processing |
| url | https://www.mdpi.com/1422-0067/24/14/11262 |
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