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. 2022 Oct;11(5):e1323.
doi: 10.1002/mbo3.1323.

DNA extraction bias is more pronounced for microbial eukaryotes than for prokaryotes

Anne Brauer  1 , Mia M Bengtsson  1   2
Affiliations

DNA extraction bias is more pronounced for microbial eukaryotes than for prokaryotes

Anne Brauer et al. Microbiologyopen. 2022 Oct.

Abstract

DNA extraction and preservation bias is a recurring topic in DNA sequencing-based microbial ecology. The different methodologies can lead to distinct outcomes, which has been demonstrated especially in studies investigating prokaryotic community composition. Eukaryotic microbes are ubiquitous, diverse, and increasingly a subject of investigation in addition to bacteria and archaea. However, little is known about how the choice of DNA preservation and extraction methodology impacts perceived eukaryotic community composition. In this study, we compared the effect of two DNA preservation methods and six DNA extraction methods on the community profiles of both eukaryotes and prokaryotes in phototrophic biofilms on seagrass (Zostera marina) leaves from the Baltic Sea. We found that, whereas both DNA preservation and extraction method caused significant bias in perceived community composition for both eukaryotes and prokaryotes, extraction bias was more pronounced for eukaryotes than for prokaryotes. In particular, soft-bodied and hard-shelled eukaryotes like nematodes and diatoms, respectively, were differentially abundant depending on the extraction method. We conclude that careful consideration of DNA preservation and extraction methodology is crucial to achieving representative community profiles of eukaryotes in marine biofilms and likely all other habitats containing diverse eukaryotic microbial communities.

Keywords: 18S rRNA; DNA extraction bias; DNA preservation; biofilm; microbial communities; seagrass microbiome.

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Conflict of interest statement

None declared.

Figures

Figure 1
Figure 1
Comparison of communities of epibiotic microbial eukaryotes (a) and prokaryotes (b) on Zostera marina treated with different DNA preservation methods and DNA extraction methods. The six different extraction methods (different shapes) that were tested are summarized and detailed in Table A1. nMDS ordinations based on Bray–Curtis distances were calculated from Hellinger transformed sequence variant counts; dashed lines indicate the 95% confidence interval of the factor preservation method. PERMANOVA results are indicated in the lower left corners, R 2 × 100 corresponds to the % of variation explained. nMDS, nonmetric multidimensional scaling; PERMANOVA, permutational multivariate analysis of variance; and rRNA, ribosomal RNA.
Figure 2
Figure 2
Significantly differentially abundant taxa (ASVs, p < 0.01 are shown) in the epibiotic microbial eukaryotic (a, c, e) and prokaryotic (b, d, f) communities on Zostera marina treated with the two different preservation (a, b) or selected DNA extraction methods (c–f) as detected by DeSeq2 parametric Wald test. Point diameter is scaled by the abundance of the ASVs. (c, d) Communities extracted using the InnuSpeed method compared to the PowerSoil method. (e, f) Communities extracted using the QuickDNA method compared to the PowerSoil method. Taxa names on arrows indicate the finest taxonomic resolution for selected ASVs. Pairwise comparisons with more than 10 significant differentially abundant taxa are shown here; see Figure A5 for the remaining comparisons
Figure A1
Figure A1
DNA yield across the different DNA preservation and extraction methods
Figure A2
Figure A2
Rarefied richness of microbial (a) eukaryotes and (b) prokaryotes across the different DNA preservation and extraction methods.
Figure A3
Figure A3
Evenness of microbial (a) eukaryotes and (b) prokaryotes across the different DNA preservation and extraction methods.
Figure A4
Figure A4
Relative abundances of the epiphytic microbial eukaryotic (a) and prokaryotic (b) taxa in the biofilm covering Zostera marina leaves
Figure A5
Figure A5
Significantly differentially abundant taxa (ASVs, p < 0.01 are shown) in the epibiotic microbial eukaryotic (a, c, e) and prokaryotic (b, d, f) communities on Zostera marina treated with different DNA extraction methods as detected by the DeSeq2 parametric Wald test. Point diameter is scaled by the abundance of the ASVs. (a, b) communities extracted by the DNAspin method compared to the PowerSoil method. (c, d) Communities extracted by the PowerBiofilm method compared to the PowerSoil method. (e, f) Communities extracted by the TotNAE method compared to the PowerSoil method. Taxa names on arrows indicate the finest taxonomic resolution for selected ASVs.
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