Best practices for methylome characterization in novel species: a case study in the microalgae Microchloropsis

Microalgae remain an important feedstock for the production of biofuels and bioproducts. Discovery of new species drives innovation for biotransformation, where bioengineering and other technological advances can significantly optimize performance. Production is predicated on deep knowledge of algal behavior predicted from genomic and phenotypic studies. However, prediction and manipulation of behavior, particularly for scale up, remains a challenge. Understanding the contribution of epigenetic processes to algal function provides another piece of this complex puzzle for achieving bioeconomy goals. Utilizing Nannochloropsis species as a model, we provide a methodological framework for investigating epigenetic processes, specifically DNA methylation, in new species and demonstrate best practices for discerning novel epigenetic modifications. We demonstrate specific forms of DNA methylation can be overlooked by traditional epigenetic analysis strategies. Using high-throughput, lower cost techniques, we provide evidence demonstrating Nannochloropsis gaditana and N. salina lack the most ubiquitous forms of eukaryotic DNA methylation (5mC and 5hmC) and instead employ N6-adenine methylation (6mA), commonly found in bacteria, in their genomes. Nannochloropsis salina and gaditana were subjected to nitrogen starvation (Replete, Starved, Deplete), or exposed to different levels of light (photoperiod light or dark). gDNA from nitrogen starved cells was treated with bisulfite and sequenced. Alterantively, Oxford Nanopore sequencing was performed for light and dark conditions and methylation calling performed using dorado and modkit.