High molecular weight DNA extraction methods lead to high quality filamentous ascomycete fungal genome assemblies using Oxford Nanopore sequencing

During the last two decades, whole-genome sequencing has revolutionized genetic research in all kingdoms, including fungi. More than 1000 fungal genomes have been submitted to sequence databases, mostly obtained through second generation short-read DNA sequencing. As a result, highly fragmented genome drafts have typically been obtained. However, with the emergence of third generation long-read DNA sequencing, the assembly challenge can be overcome and highly contiguous assemblies obtained. Such attractive results, however, are extremely dependent on the ability to extract highly purified high molecular weight (HMW) DNA. Extraction of such DNA is currently a significant challenge for all species with cell walls, not least fungi. In this study, four isolates of filamentous ascomycetes (Apiospora pterospermum, Aspergillus sp. (subgen. Cremei), Aspergillus westerdijkiae, and Penicillium aurantiogriseum) were used to develop extraction and purification methods that result in HMW DNA suitable for third generation sequencing. We have tested and propose two straightforward extraction methods based on treatment with either a commercial kit or traditional phenol-chloroform extraction both in combination with a single commercial purification method that result in high quality HMW DNA from filamentous ascomycetes. Our results demonstrated that using these DNA extraction methods and coverage, above 75x of our haploid filamentous ascomycete fungal genomes result in complete and contiguous assemblies.

Automated summary

In the past two decades, whole-genome sequencing has greatly impacted genetic research in various kingdoms, including fungi. With the advancement of third-generation long-read DNA sequencing, highly contiguous assemblies can be obtained, but the challenge lies in extracting high molecular weight DNA, particularly from species with cell walls like fungi. Developing efficient extraction and purification methods is crucial for obtaining quality DNA for sequencing, and the study successfully demonstrated two straightforward methods for extracting high quality, high molecular weight DNA from filamentous ascomycetes, resulting in complete and contiguous assemblies for haploid fungal genomes.