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Updated: Nov 8, 2025
Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells
Published on: April 4, 2016
An improved direct metamobilome approach increases the detection of larger-sized circular elements across kingdoms
Katrine Wacenius Skov Alanin1, Tue Sparholt Jørgensen2, Patrick Denis Browne1
1Department of Environmental Science, Aarhus University, Roskilde, Denmark; Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmark.
Abstract:
Mobile genetic elements (MGEs) are instrumental in natural prokaryotic genome editing, permitting genome plasticity and allowing microbes to accumulate genetic diversity. MGEs serve as a vast communal gene pool and include DNA elements such as plasmids and bacteriophages (phages) among others. These mobile DNA elements represent a human health risk as they can introduce new traits, such as antibiotic resistance or virulence, to a bacterial strain. Sequencing libraries targeting environmental circular MGEs, referred to as metamobilomes, may broaden our current understanding of the mechanisms behind the mobility, prevalence and content of these elements. However, metamobilomics is affected by a severe bias towards small circular elements, introduced by multiple displacement amplification (MDA). MDA is typically used to overcome limiting DNA quantities after the removal of non-circular DNA during library preparations. By examining the relationship between sequencing coverage and the size of circular MGEs in paired metamobilome datasets with and without MDA, we show that larger circular elements are lost when using MDA. This study is the first to systematically demonstrate that MDA is detrimental to detecting larger-sized plasmids if small plasmids are present. It is also the first to show that MDA can be omitted when using enzyme-based DNA fragmentation and PCR in library preparation kits such as Nextera XT® from Illumina.
Insights
Multiple displacement amplification (MDA) hinders the detection of large plasmids in prokaryotic metamobilome studies. Omitting MDA is recommended for accurate analysis of mobile genetic elements (MGEs) and their impact on microbial genomes.
Area of Science:
- Microbiology
- Genomics
- Molecular Biology
Background:
- Mobile genetic elements (MGEs), including plasmids and bacteriophages, drive prokaryotic genome evolution and plasticity.
- MGEs contribute to the spread of critical traits like antibiotic resistance and virulence, posing risks to human health.
- Metamobilomics, the study of environmental circular MGEs, offers insights into MGE mobility, prevalence, and genetic content.
Purpose of the Study:
- To investigate the impact of multiple displacement amplification (MDA) on the detection of circular MGEs in metamobilome datasets.
- To systematically evaluate the bias introduced by MDA towards smaller circular elements.
- To determine if MDA can be omitted in library preparation for accurate MGE detection.
Main Methods:
- Comparison of paired metamobilome datasets generated with and without MDA.
- Analysis of the relationship between sequencing coverage and the size of circular MGEs.
- Evaluation of library preparation kits, including those using enzyme-based DNA fragmentation (e.g., Nextera XT®).
Main Results:
- MDA significantly biases metamobilomics towards smaller circular MGEs, leading to the loss of larger circular elements.
- This study provides the first systematic evidence that MDA is detrimental to detecting large plasmids in the presence of small plasmids.
- MDA can be omitted when employing enzyme-based DNA fragmentation and PCR in library preparation kits.
Conclusions:
- MDA introduces a critical bias in metamobilomics, underrepresenting larger plasmids and potentially skewing our understanding of MGE dynamics.
- Researchers should consider omitting MDA in library preparation protocols, especially when using enzyme-based fragmentation, to improve the detection of diverse MGEs.
- Avoiding MDA is crucial for a more comprehensive analysis of MGEs, their role in microbial adaptation, and their implications for public health.
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