Abstract
As technologies advance alongside metabarcoding and metagenomic resources, particularly for larger fungal genomes, DNA extraction methods must be optimized to meet higher thresholds, especially from complex environmental substrates. This study focused on extracting fungal genomic compounds from woody substrates, a challenge due to the embedment of endophytic and saprotrophic fungi within wood cells, the physical recalcitrance of wood, the adsorption of nucleic acids to wood polymers, and the release of downstream inhibitors. Hypothesizing that cetyltrimethylammonium bromide would be the best option, we compared prominent methods by extracting and sequencing microbial DNA from sound and decayed birch (Betula papyrifera) and pine (Pinus resinosa). DNA quantities varied significantly depending on extraction methods and decay stage. The quality of DNA, in terms of purity and integrity, significantly impacted whether the samples could be amplified and sequenced. However, amplicon sequencing of bacterial and fungal communities revealed no significant extraction bias. This, along with the sequencing effectiveness and cost/time efficiency, indicates that Qiagen is the gold standard for woody substrates. This study increases confidence in published amplicon data sets regardless of the extraction methods, provides a cost-benefit table for making protocol decisions, and offers guidance on fungal DNA extractions from complex organic substrates (sound and decayed wood) that would best suit future metagenomic efforts.