Journal
ANAEROBE
Volume 38, Issue -, Pages 39-46Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.anaerobe.2015.11.008
Keywords
Anaerobic fungi; Cryopreservation; Genome isolation; Gut fungi; Next generation sequencing; Microbiome; Genome sequencing; Non-model microbes
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Funding
- Office of Science (BER), U.S. Department of Energy [DE-SC0010352]
- Institute for Collaborative Biotechnologies [W911NF-09-0001]
- Mellichamp Academic Initiative in Sustainability at UC Santa Barbara
- Office of Biological and Environmental Research [DE-AC02-05CH11231, DE-AC05-76RL01830]
- EPSRC [EP/M007359/1] Funding Source: UKRI
- Engineering and Physical Sciences Research Council [EP/M007359/1] Funding Source: researchfish
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Cell storage and DNA isolation are essential to developing an expanded suite of microorganisms for biotechnology. However, many features of non-model microbes, such as an anaerobic lifestyle and rigid cell wall, present formidable challenges to creating strain repositories and extracting high quality genomic DNA. Here, we establish accessible, high efficiency, and robust techniques to store lignocellulolytic anaerobic gut fungi long term without specialized equipment. Using glycerol as a cryoprotectant, gut fungal isolates were preserved for a minimum of 23 months at -80 degrees C. Unlike previously reported approaches, this improved protocol is non-toxic and rapid, with samples surviving twice as long with negligible growth impact. Genomic DNA extraction for these isolates was optimized to yield samples compatible with next generation sequencing platforms (e.g. Illumina, PacBio). Popular DNA isolation kits and precipitation protocols yielded preps that were unsuitable for sequencing due to carbohydrate contaminants from the chitin-rich cell wall and extensive energy reserves of gut fungi. To address this, we identified a proprietary method optimized for hardy plant samples that rapidly yielded DNA fragments in excess of 10 kb with minimal RNA, protein or carbohydrate contamination. Collectively, these techniques serve as fundamental tools to manipulate powerful biomass-degrading gut fungi and improve their accessibility among researchers. (C) 2015 Elsevier Ltd. All rights reserved.
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