Tools and techniquesHigh molecular weight genomic DNA mini-prep for filamentous fungi

Purification of high quality genomic DNA (gDNA) from filamentous fungi suitable for whole genome sequencing has previously involved many steps. Here, we report a simple and easy-to-follow mini-preparation protocol for high molecular weight (∼20 kb) gDNA from filamentous fungi including Aspergillus and Eurotium. This comprehensive protocol includes graphic step-by-step instructions for inoculation, homogenization, and purification of gDNA. The most critical step is a thorough 3-5 min homogenization of the freeze-dried mycelium using a motorized hand-held homogenizer with a mini spatula inserted. Approximately 20 mg of the fine mycelial powder is then subjected to a modified procedure for the DNeasy Plant Mini Kit (Qiagen). This Qiagen spin column protocol avoids precipitation, dryness, and resuspension of gDNA, which can cause shearing and loss of gDNA. Final gDNA yields from ∼20 mg of fine mycelial powder are 8 to 20 μg with a consistent 260/280 nm absorbance ratio of ∼1.9. All 30 gDNA samples we purified using our method were of high molecular weight (∼20 kb). Whole genome sequencing of these DNA samples resulted in 160-260 X coverage with 2 × 150 reads using NextSeq 500. These gDNAs are also of a suitable quality for Southern blotting and PCR-based amplification of various genes in filamentous fungi.