Boycheva, SRoberton, AMPisaniello, APardesi, BWhite, WLClements, KD2024-11-062024-11-062024-10-09Anaerobe, ISSN: 1075-9964 (Print); 1095-8274 (Online), Elsevier BV, 90, 102915-. doi: 10.1016/j.anaerobe.2024.1029151075-99641095-8274http://hdl.handle.net/10292/18230Objectives: The central challenge in third-generation sequencing lies in meeting the requirements for DNA quality (integrity and purity) and quantity. Therefore, novel improvements in DNA extraction methods are needed to satisfy these requirements. We reasoned that in anaerobic microbial communities, the presence of certain strict anaerobes containing oxygen-activated DNase activity might contribute substantially to the poor integrity of extracted metagenomic DNA (or genomic DNA from some pure cultures) if exposed to air. Methods: To test this hypothesis, we developed an enhanced genomic and metagenomic DNA isolation technique that we applied to a specifically chosen set of both strict and aerotolerant anaerobes, as well as to the hindgut microbiota of a herbivorous marine fish. Results: Considering the quality (or degradation) of extracted DNA obtained under anaerobic versus aerobic conditions, we found that DNA extracted aerobically from cells of some strict anaerobes showed more degradation of high molecular weight DNA than analogous preparations under anaerobic conditions. In contrast, with the selected aerotolerant anaerobes, no discernible difference was found between the molecular sizes of DNA extracted aerobically and anaerobically. Metagenomic DNA extracted from the fish hindgut microbiota showed higher yields and better quality under anaerobic conditions compared to aerobic conditions. Conclusion: Our study effectively demonstrates the advantages of our improved extraction protocol in anaerobic conditions. This is evident through the improved quality of extracted DNA. Such findings may be valuable for studies, especially metagenomic studies, where the quality and quantity of DNA are crucial for downstream analysis.© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0http://creativecommons.org/licenses/by-nc/4.0/Anaerobic conditionsDNA degradationDNA extractionHigh-molecular-weight DNAMetagenomic DNAOxygen-activated DNase3107 Microbiology31 Biological SciencesGeneticsHuman Genome0605 Microbiology1108 Medical MicrobiologyMicrobiology3107 Microbiology3202 Clinical sciencesJournal ArticleOpenAccess10.1016/j.anaerobe.2024.102915