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Tissue Cultivation, Preparation, and Extraction of High Molecular Weight DNA for Single-Molecule Genome Sequencing of Plant-Associated Fungi

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Microbial Environmental Genomics (MEG)

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2605))

Abstract

Extraction of high-quality, high molecular weight DNA is a critical step for sequencing an organism’s genome. For fungi, DNA extraction is often complicated by co-precipitation of secondary metabolites, the most destructive being polysaccharides, polyphenols, and melanin. Different DNA extraction protocols and clean-up methods have been developed to address challenging materials and contaminants; however, the method of fungal cultivation and tissue preparation also plays a critical role to limit the production of inhibitory compounds prior to extraction. Here, we provide protocols and guidelines for (i) fungal tissue cultivation and processing with solid media containing a cellophane overlay or in liquid media, (ii) DNA extraction with customized recommendations for taxonomically and ecologically diverse plant-associated fungi, and (iii) assessing DNA quantity and quality for downstream genome sequencing with single-molecule technology such as PacBio.

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Acknowledgements

We would like to thank Jason Stajich, Rytas Vilgalys, David Kudrna, Marc Orbach, and Ali Mandel for the helpful discussion on fungal DNA extraction methods. The research of AK and LF was funded by the Labex ARBRE (ANR-11-LABX-0002-01). The work conducted by the U.S. Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy operated under Contract No. DE-AC02-05CH11231.

Author Contributions

Authors are ordered by scientific role: laboratory technicians are listed first, in alphabetical order (LF, MK, LM, and YY), followed by senior authors, in alphabetical order (KB, AK, JMU). Specific protocols were contributed by the following authors (fruiting body collection: AK and LF; cellophane overlay and liquid cultures: JMU and LPM, with input from AK and LF; DNA extraction: AK, LF, JMU, and LPM; DNA quality control: KB, YY, and MK).

Author information

Authors and Affiliations

  1. Université de Lorraine, INRAE, UMR1136 Interactions Arbres/Microorganismes, Centre INRAE Grand Est-Nancy, Champenoux, France

    Laure Fauchery & Annegret Kohler

  2. Department of Energy, The Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Maxim Koriabine

  3. BIO5 Institute and Department of Biosystems Engineering, The University of Arizona, Tucson, AZ, USA

    Lillian P. Moore, Yuko Yoshinaga, Kerrie Barry & Jana M. U’Ren

Authors
  1. Laure Fauchery
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  2. Maxim Koriabine
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  3. Lillian P. Moore
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  4. Yuko Yoshinaga
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  5. Kerrie Barry
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  6. Annegret Kohler
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  7. Jana M. U’Ren
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Corresponding authors

Correspondence to Kerrie Barry , Annegret Kohler or Jana M. U’Ren .

Editor information

Editors and Affiliations

  1. Tree-Microbe Interact (IAM); Biogeochem, UMR1136 INRA/University of Lorraine, Labex ARB, Champenoux, France

    Francis Martin

  2. Université de Lorraine, INRAE, UMR1136 “Interactions Arbres-Microorganismes”, F-54280 Champenoux, France

    Stephane Uroz

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Fauchery, L. et al. (2023). Tissue Cultivation, Preparation, and Extraction of High Molecular Weight DNA for Single-Molecule Genome Sequencing of Plant-Associated Fungi. In: Martin, F., Uroz, S. (eds) Microbial Environmental Genomics (MEG). Methods in Molecular Biology, vol 2605. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2871-3_5

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  • DOI: https://doi.org/10.1007/978-1-0716-2871-3_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2870-6

  • Online ISBN: 978-1-0716-2871-3

  • eBook Packages: Springer Protocols

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