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Open AccessArticle

Evaluation of Sequencing Library Preparation Protocols for Viral Metagenomic Analysis from Pristine Aquifer Groundwaters

Helmholtz Centre for Environmental Research - UFZ, Department of Environmental Microbiology, 04318 Leipzig, Germany
Friedrich Schiller University Jena, RNA Bioinformatics and High-Throughput Analysis, 07743 Jena, Germany
European Virus Bioinformatics Center, 07743 Jena, Germany
Bioinformatics Group, Department of Computer Science and Interdisciplinary Center for Bioinformatics, University Leipzig, 04081 Leipzig, Germany
German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, 04103 Leipzig, Germany
Author to whom correspondence should be addressed.
Viruses 2019, 11(6), 484;
Received: 31 March 2019 / Revised: 26 May 2019 / Accepted: 27 May 2019 / Published: 28 May 2019
(This article belongs to the Special Issue Virus Bioinformatics)
Viral ecology of terrestrial habitats is yet-to be extensively explored, in particular the terrestrial subsurface. One problem in obtaining viral sequences from groundwater aquifer samples is the relatively low amount of virus particles. As a result, the amount of extracted DNA may not be sufficient for direct sequencing of such samples. Here we compared three DNA amplification methods to enrich viral DNA from three pristine limestone aquifer assemblages of the Hainich Critical Zone Exploratory to evaluate potential bias created by the different amplification methods as determined by viral metagenomics. Linker amplification shotgun libraries resulted in lowest redundancy among the sequencing reads and showed the highest diversity, while multiple displacement amplification produced the highest number of contigs with the longest average contig size, suggesting a combination of these two methods is suitable for the successful enrichment of viral DNA from pristine groundwater samples. In total, we identified 27,173, 5,886 and 32,613 viral contigs from the three samples from which 11.92 to 18.65% could be assigned to taxonomy using blast. Among these, members of the Caudovirales order were the most abundant group (52.20 to 69.12%) dominated by Myoviridae and Siphoviridae. Those, and the high number of unknown viral sequences, substantially expand the known virosphere. View Full-Text
Keywords: viral metagenome; groundwater; aquifer; AquaDiva; sequencing library preparation viral metagenome; groundwater; aquifer; AquaDiva; sequencing library preparation
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Kallies, R.; Hölzer, M.; Brizola Toscan, R.; Nunes da Rocha, U.; Anders, J.; Marz, M.; Chatzinotas, A. Evaluation of Sequencing Library Preparation Protocols for Viral Metagenomic Analysis from Pristine Aquifer Groundwaters. Viruses 2019, 11, 484.

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