DNA methylation signature detection using ultra-rapid, long-read nanopore genome sequencing

Abstract Mendelian disorders of epigenetic machinery (MDEMs) are often clinically characterised by neurodevelopmental disorders (NDDs) and molecularly by disorder-specific genome-wide DNA methylation (DNAm) patterns, which are called DNAm signatures. DNAm signatures are used for the diagnostics of MDEMs, re-classification of variants of uncertain significance, and studying underlying molecular mechanisms. To date, ~100 DNAm signatures have been identified, mostly by utilising methylation microarrays, limiting analysis to only ~3% of known CpG sites. We aimed to utilize Oxford Nanopore longread genome sequencing (LR-GS) as a one-test-fits-all tool for detection of genetic variants, as well as DNA methylation profiles. We present preliminary results showing that nanopore long-read sequencing can be used to differentiate individuals with Kabuki and Kleefstra syndromes from controls using previously published array-based DNAm signatures: 1) on hierarchical clustering; 2) PCA analysis, and 3) SVM classifier, publicly available on EpigenCentral (optimised for the Oxford Nanopore DNAm data input). Importantly, LR-GS also identified the causative variants in these individuals. The classification remains robust with downsampling coverage up to 10–15x, while performance varies with lower coverage. By utilizing this approach, we confirmed CHARGE syndrome in an affected newborn from ICU enrolled in our ultrarapid Oxford Nanopore LR-GS project, identifying the respective DNAm signature before variant calling and, subsequently, the causative variant. As the next step, we plan to derive the DNAm signatures utilizing whole-genome DNAm data to better understand the underlying disease mechanisms. In summary, we show that DNAm signatures can be robustly applied to Oxford Nanopore LR-GS, impacting ultrarapid diagnostics and clinical care. Biography Dmitrijs Rots obtained his MD degree in Riga, Latvia, and continued with PhD studies in Radboudumc, Nijmegen, Netherlands, at the group of Prof. T. Kleefstra, working on clinical, molecular, and DNA methylation features of Mendelian neurodevelopment disorders of epigenetic machinery. Currently, he continues as a post-doc at the group of Prof. T. Kleefstra at Erasmus MC, Rotterdam, where he is involved in the implementation of nanopore sequencing with a focus on DNA methylation and its applications.