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Amar, Y. ; Lagkouvardos, I.* ; Silva, R.L. ; Ishola, O.A. ; Fösel, B. ; Kublik, S. ; Schöler, A. ; Niedermeier, S.* ; Bleuel, R. ; Zink, A. ; Neuhaus, K.* ; Schloter, M. ; Biedermann, T. ; Köberle, M.*

Pre-digest of unprotected DNA by Benzonase improves the representation of living skin bacteria and efficiently depletes host DNA.

Microbiome 9:123 (2021)
Publ. Version/Full Text Research data DOI PMC
Open Access Gold
Creative Commons Lizenzvertrag
Background: The identification of microbiota based on next-generation sequencing (NGS) of extracted DNA has drastically improved our understanding of the role of microbial communities in health and disease. However, DNA-based microbiome analysis cannot per se differentiate between living and dead microorganisms. In environments such as the skin, host defense mechanisms including antimicrobial peptides and low cutaneous pH result in a high microbial turnover, likely resulting in high numbers of dead cells present and releasing substantial amounts of microbial DNA. NGS analyses may thus lead to inaccurate estimations of microbiome structures and consequently functional capacities. Results: We investigated in this study the feasibility of a Benzonase-based approach (BDA) to pre-digest unprotected DNA, i.e., of dead microbial cells, as a method to overcome these limitations, thus offering a more accurate assessment of the living microbiome. A skin mock community as well as skin microbiome samples were analyzed using 16S rRNA gene sequencing and metagenomics sequencing after DNA extraction with and without a Benzonase digest to assess bacterial diversity patterns. The BDA method resulted in less reads from dead bacteria both in the skin mock community and skin swabs spiked with either heat-inactivated bacteria or bacterial-free DNA. This approach also efficiently depleted host DNA reads in samples with high human-to-microbial DNA ratios, with no obvious impact on the microbiome profile. We further observed that low biomass samples generate an α-diversity bias when the bacterial load is lower than 10 CFU and that Benzonase digest is not sufficient to overcome this bias. Conclusions: The BDA approach enables both a better assessment of the living microbiota and depletion of host DNA reads. [MediaObject not available: see fulltext.] Graphical abstract: [Figure not available: see fulltext.]
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Publication type Article: Journal article
Document type Scientific Article
Corresponding Author
Keywords 16s Rrna ; Benzonase ; Diversity ; Dna Extraction ; Live/dead ; Low Biomass ; Next-generation Sequencing ; Skin ; Skin Microbiome; Ethidium Monoazide; Propionibacterium-acnes; Microbiome; Dead; Contamination; Pcr; Diversity; Strain; Live; Extraction
ISSN (print) / ISBN 2049-2618
e-ISSN 2049-2618
Journal Microbiome
Quellenangaben Volume: 9, Issue: 1, Pages: , Article Number: 123 Supplement: ,
Publisher BioMed Central
Publishing Place London
Non-patent literature Publications
Reviewing status Peer reviewed
Institute(s) Unit for Clinical Allergology (KKG-EKA)
Research Unit Comparative Microbiome Analysis (COMI)
Grants Forschungskuratorium Textil, Bundesministerium für Wirtschaft und Energie
Fonds national de la Recherche Luxembourg
Helmholtz-Gemeinschaft
Deutsche Forschungsgemeinschaft