Isolation of High-Molecular-Weight (HMW ) DNA from Fusarium oxysporum for Long-Read Sequencing.

In many instances Fusarium oxysporum genomes are complex and challenging to assemble mainly due to the increased number of repetitive elements and variable numbers of supernumerary chromosomes, which are primarily associated with pathogenicity. Hence, to obtain the accurate F. oxysporum genome assembly and high-resolution sequence information, protocols for versatile, reliable, and high recovery of high-quality DNA for diverse sequencing platforms are instrumental. Here, we describe two protocols for the isolation of DNA from isolates of F. oxysporum. One is a quick and easy method for high-throughput extraction of DNA to rapidly screen diverse isolates by marker-assisted PCR analysis. Another is to harvest high-quality and high-molecular-weight DNA for whole-genome sequencing. In addition, we also include a library preparation protocol optimized for the third-generation sequencing technology using the portable MinION device to obtain long-read sequences. These protocols can be potentially further applied for all Fusarium spp. and other fungal pathogens, including Verticillium.