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Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition

Berith E Knudsen, Lasse Bergmark, Patrick Munk, Oksana Lukjancenko, Anders Prieme, Frank M Aarestrup, View ORCID ProfileSunje Johanna Pamp
doi: https://doi.org/10.1101/064394
Berith E Knudsen
Technical University of Denmark;
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Lasse Bergmark
Technical University of Denmark;
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Patrick Munk
Technical University of Denmark;
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Oksana Lukjancenko
Technical University of Denmark;
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Anders Prieme
University of Copenhagen
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Frank M Aarestrup
Technical University of Denmark;
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Sunje Johanna Pamp
Technical University of Denmark;
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  • ORCID record for Sunje Johanna Pamp
  • For correspondence: sjpa@food.dtu.dk
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Abstract

Explorations of complex microbiomes using genomics greatly enhance our understanding about their diversity, biogeography, and function. The isolation of DNA from microbiome specimens is a key prerequisite for such examinations, but challenges remain in obtaining sufficient DNA quantities required for certain sequencing approaches, achieving accurate genomic inference of microbiome composition, and facilitating comparability of findings across specimen types and sequencing projects. These aspects are particularly relevant for the genomics-based global surveillance of infectious agents and antimicrobial resistance from different reservoirs. Here, we compare a total of eight DNA extraction procedures for three specimen types (human feces, pig feces, hospital sewage), assess DNA extraction using spike-in controls and different types of beads for bead-beating facilitating cell lysis. We evaluate DNA concentration, purity, and stability, and microbial community composition using 16S rRNA gene sequencing and for selected samples using shotgun metagenomic sequencing. Our results suggest that inferred community composition was dependent on inherent specimen properties as well as DNA extraction method. We further show that bead-beating or enzymatic treatment can increase the extraction of DNA from Gram-positive bacteria. Final DNA quantities could be increased by isolating DNA from a larger volume of cell lysate compared to standard protocols. Based on this insight, we have designed an improved DNA isolation procedure optimized for microbiome genomics that can be used for the three examined specimen types and potentially also for other biological specimens.

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The copyright holder for this preprint is the author/funder. It is made available under a CC-BY 4.0 International license.
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  • Posted July 18, 2016.

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Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition
Berith E Knudsen, Lasse Bergmark, Patrick Munk, Oksana Lukjancenko, Anders Prieme, Frank M Aarestrup, Sunje Johanna Pamp
bioRxiv 064394; doi: https://doi.org/10.1101/064394
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Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition
Berith E Knudsen, Lasse Bergmark, Patrick Munk, Oksana Lukjancenko, Anders Prieme, Frank M Aarestrup, Sunje Johanna Pamp
bioRxiv 064394; doi: https://doi.org/10.1101/064394

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