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

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.

Identifying Eukaryotes and Factors Influencing Their Biogeography in Drinking Water Metagenomes

“…In any case, while commercial extraction methods were shown to be able to successfully extract eukaryotic DNA, , specific processing techniques or the use of dedicated enzymes might further increase yields and/or quality. However, the variety of eukaryotic phenotypes (e.g., soft-shelled, hard-shelled) exacerbates the extraction bias, making it unlikely that a single optimal extraction method could be developed . At last, the sequencing depth affects the ability to recover rarer taxa .…”

“…However, the variety of eukaryotic phenotypes (e.g., soft-shelled, hard-shelled) exacerbates the extraction bias, making it unlikely that a single optimal extraction method could be developed. 96 At last, the sequencing depth affects the ability to recover rarer taxa. 97 efforts did not lead to a significant increase in eukaryotic fractions (p-value = 0.38) due to the confounding effect caused by the different complexity of the microbial community in each sample.…”

Identifying eukaryotes in drinking water metagenomes and factors influencing their biogeography

“…In any case, while commercial extraction methods were shown to be able to successfully extract eukaryotic DNA 87,88 , specific processing techniques 89 or the use of dedicated enzymes 90 might further increase yields and/or quality. Noteworthy, the variety of eukaryotic phenotypes (e.g., soft-shelled, hard-shelled) complicates the ideation of a single optimal extraction method and possibly exacerbates the extraction bias 91 . Lastly, sequencing depth affects the ability to recover rarer taxa 92 .…”

Microbial diversity and abundance vary along salinity, oxygen, and particle size gradients in the Chesapeake Bay

“…DNA Extraction Efficiency : Our assumption of 100% DNA extraction efficiency (Equation ()). may lead to underestimates of the abundance of some taxa (Brauer & Bengtsson, 2022; Han et al, 2018; Nearing et al, 2021). Adding spikes before DNA extraction (following Gifford et al, 2020) could correct for this loss, but this was not done here.…”