Third generation sequencing paves the way for rapid and complete fungal genome sequening

Verticillium dahliae, causal agent of vascular wilt disease, is one of the most notorious plant pathogens on tomato. By using population genome sequencing with the Illumina platform, we have recently shown that frequent genomic rearrangements drive the evolution of lineage-specific regions that establish virulence and niche adaptation in this species. Due to limitations associated with second generation sequencing, in particular the short read length and lack of coverage of repetitive sequence stretches, the exact genomic signatures at the site of the chromosomal rearrangements, and thus the molecular mechanism that establishes these modifications, remain largely unknown. In order to obtain detailed genomic information on the recombination sites, we re-sequenced the ~37 Mb genome of V. dahliae strain JR2 using single-molecule real time (SMRT) sequencing with PacBio technology. To this end, we generated ~8 Gb of sequencing data. De novo assembly was performed resulting in 66 contigs of which 16 covered 99% of the complete genome as inferred from an optical map. Subsequent manual and software-guided scaffolding resulted in a gapless assembly of all eight complete chromosomes. Thus, the re-assembled V. dahliae strain JR2 genome represents the first finished, gapless fungal genome. We subsequently applied comparative genomics and inferred the exact positions of the previously identified genomic rearrangements. Our approach to identify genomic signatures at the recombination sites will be discussed. This study highlights the superior genome assembly qualities of third generation sequencing technologies and exemplifies the pivotal role of a finished genome in understanding the evolution and biology of fungal pathogens. We anticipate that third generation sequencing will pave the way for rapid and affordable genome sequencing approaches aiming for finished assemblies in fungal and also other eukaryotic species.