Nanopore methylation detection in the non-model organism Verticillium dahliae

Abstract

The DNA of an organism encodes the blueprint necessary for all cellular functions. However, the expression of this code varies in each cell. This variation is, in part, attributed to DNA methylation. Which is a modification of the DNA where methyl groups are added on top of it. These modifications influence the expression and regulation of the genetic code. DNA methylation has been studied in a limited number of fungi, usually through bisulfite sequencing. Nanopore sequencing is a third-generation sequencing method that can detect methylation through the use of deep learning models. Thus far, it has not been studied how well these models apply to fungi. In this study, we investigated Nanopore methylation detection in Verticillium dahliae by comparing it to bisulfite sequencing. Verticillium dahliae is a plant-pathogen that infects over 400 plant species and causes significant crop loses. Both bisulfite and Nanopore tools are capable of detecting 5mC methylation. Analysis at the global level shows agreement between different Nanopore tools and bisulfite data, with Nanopore demonstrating higher levels of methylation over longer stretches of repeating areas in the genome. Nanopore basecalls from different tools have similar quality but were not shown to agree with each other or with bisulfite data on the exact locations of methylated cytosines. An analysis of a human Nanopore sample showed a significant improvement in local methylated cytosine overlap between Nanopore tools. Overall, our study demonstrates that various Nanopore tools and bisulfite data align on the location of methylation when observed from a global perspective, with Nanopore tools having the advantage. However, discrepancies arise among Nanopore tools themselves and when compared to bisulfite data at the level of specific cytosines. Subsequent analysis of Nanopore tools with human data suggests that Nanopore models may not be sufficiently generalized for precise methylation position detection in fungi. As such, Nanopore currently cannot be used to analyse exact methylated cytosine locations in fungi. This reveals a need for tools designed specifically for methylation detection in fungi.