Long-read Sequencing of Fungal DNA

To prepare long-chain genomic DNA isolated from JCM 15448, DNA extraction was performed as described previously [49 (link)]. Briefly, fungal cells were cultured in 10 ml RPMI1640 medium for 2 days, suspended in 450 μl Tris-EDTA (TE) buffer. The cell suspension was supplemented with 50 μl SDS and 500 μl phenol/chloroform, followed by bead-beating for 10 min by vortexing. After centrifugation at 17,400 x g for 5 min, the upper aqueous phase was subjected to electrophoresis on a 1% TAE agarose gel, followed by purification of long size DNA (15-40kb) using a Zymoclean large-fragment DNA recovery kit (Zymoresearch, Irvine, CA, USA). Short read and long read DNA libraries with purified DNA were constructed using QIAseq FX DNA Library Kit (QIAGEN, Hilden, Germany) and the SMRTbell template prep kit 1.0 (PacBio, Menlo Park, CA, USA) according to the manufacturer instructions, followed by whole-genome sequencing using NextSeq (Illumina, San Diego, CA, USA), MiSeq (Illumina), and Sequel (PacBio).