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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 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 Blautia, Faecalibacterium, and Parabacteroides. 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.

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Document Type:Article
Author:Claudio Neidhöfer, Maria Bagniceva, Nina Wetzig, Martin A. Sieber, Ralf Thiele, Marijo Parčina
Parent Title (English):International Journal of Molecular Sciences
Article Number:11262
Number of pages:10
Publishing Institution:Hochschule Bonn-Rhein-Sieg
Date of first publication:2023/07/09
Copyright:© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license
Keyword:DNA extraction protocols; automation of sample processing; extraction-linked bias; human microbiome; microbial community structure; microbiome analyses
Departments, institutes and facilities:Fachbereich Informatik
Fachbereich Angewandte Naturwissenschaften
Institut für funktionale Gen-Analytik (IFGA)
Dewey Decimal Classification (DDC):5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 576 Genetik und Evolution
Entry in this database:2023/07/11
Licence (German):License LogoCreative Commons - CC BY - Namensnennung 4.0 International