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).
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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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