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Title: Robust and effective methodologies for cryopreservation and DNA extraction from anaerobic gut fungi

Abstract

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. 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 °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 accessibilitymore » among researchers.« less

Authors:
ORCiD logo [1];  [1];  [2];  [1]
  1. Univ. of California, Santa Barbara, CA (United States). Dept. of Chemical Engineering
  2. Harper Adams Univ., Newport (United Kingdom). Animal Production, Welfare and Veterinary Sciences
Publication Date:
Research Org.:
Univ. of California, Santa Barbara, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER); US Army Research Office (ARO)
OSTI Identifier:
1485178
Alternate Identifier(s):
OSTI ID: 1398088
Grant/Contract Number:  
SC0010352; AC02-05CH11231; AC05-76RL01830; W911NF-09-0001
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Anaerobe
Additional Journal Information:
Journal Volume: 38; Journal ID: ISSN 1075-9964
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; anaerobic fungi; cryopreservation; genome isolation; gut fungi; next generation sequencing; microbiome; genome sequencing; non-model microbes

Citation Formats

Solomon, Kevin V., Henske, John K., Theodorou, Michael K., and O'Malley, Michelle A. Robust and effective methodologies for cryopreservation and DNA extraction from anaerobic gut fungi. United States: N. p., 2015. Web. doi:10.1016/j.anaerobe.2015.11.008.
Solomon, Kevin V., Henske, John K., Theodorou, Michael K., & O'Malley, Michelle A. Robust and effective methodologies for cryopreservation and DNA extraction from anaerobic gut fungi. United States. doi:10.1016/j.anaerobe.2015.11.008.
Solomon, Kevin V., Henske, John K., Theodorou, Michael K., and O'Malley, Michelle A. Tue . "Robust and effective methodologies for cryopreservation and DNA extraction from anaerobic gut fungi". United States. doi:10.1016/j.anaerobe.2015.11.008. https://www.osti.gov/servlets/purl/1485178.
@article{osti_1485178,
title = {Robust and effective methodologies for cryopreservation and DNA extraction from anaerobic gut fungi},
author = {Solomon, Kevin V. and Henske, John K. and Theodorou, Michael K. and O'Malley, Michelle A.},
abstractNote = {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. 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 °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.},
doi = {10.1016/j.anaerobe.2015.11.008},
journal = {Anaerobe},
issn = {1075-9964},
number = ,
volume = 38,
place = {United States},
year = {2015},
month = {11}
}

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