High Molecular Weight Genomic DNA Extraction

QM6a was inoculated on petri-plates with malt extract agar (MEA) medium at 25 °C until full asexual sporulation was observed (~5 days). 2 × 10⁸ conidial spores were collected, and then inoculated in 50 mL potato dextrose medium (PDB) at 30 °C for 6 h. The germinated hyphae were harvested by centrifugation at 3000g for 5 min at room temperature and incubated in 2 mL lysing enzyme buffer [0.1 M KH₂PO₄ (pH 5), 1.2 M Sorbitol, 5% lysing enzyme (Sigma, USA)] at 30 °C for 1.5 h. The protoplasts were harvested by centrifugation at 600g for 10 min at 4 °C, dissolved in 1.2 mL GuHCl solution (43% guanidine-HCl, 0.1 M EDTA pH 8.0, 0.15 M NaCl, 0.05% Sarkosyl) at 65 °C for 20 min and then mixed with 6.4 mL ice-cold ethanol to precipitate the genomic DNA. The pellet was dissolved in 10× TE with 0.6 mg/mL RNase H at 37 °C for 1 h and then in 0.4 mg/mL proteinase K at 65 °C for 1 h. The genomic DNA was purified with the phenol:chloroform:isoamyl alcohol (25:24:1) method and then recovered by standard precipitation with ethanol. Next, the quality of high molecular weight genomic DNA for Illumina MiSeq and PacBio sequencing was validated by PFGE. The genomic DNA was separated in a 1% agarose gel in 0.5× TBE buffer, using a CHEF DR II (Biorad) with 0.5× TBE running buffer, continuously refrigerated at 14 °C and 6 V/cm (current 110–125 mA) for 18 h. The Lambda DNA-Mono Cut Mix (New England Biolabs, N3019S) was used as size marker. Visualization was performed after staining with ethidium bromide after the electrophoresis.