Andrey Eremin

Circulating cell-free DNA (cfDNA) enables minimally invasive assessment of chromatin organization and DNA modifications. Whether such information can be reliably recovered under conditions of limited plasma input (below 1 mL) and ultra-low sequencing depth remains unclear. We performed low-pass whole-genome Oxford Nanopore sequencing (down to 0.01× coverage) of plasma cfDNA from young and elderly donors and jointly analyzed fragment length distributions and base modifications (5mC, 5hmC, 6mA). In parallel, we analyzed an enzymatically fragmented model DNA system to assess whether controlled in vitro fragmentation can reproduce cfDNA-like nucleosomal profiles and associated modification patterns. Despite shallow coverage, cfDNA samples displayed reproducible mono-, di-, tri-, and tetra-nucleosomal peaks, indicating that major fragmentomic features can be retained under ultra-low-coverage conditions. Modification-aware basecalling enabled exploratory quantification of global modification fractions across nucleosomal size classes and nomination of candidate group-specific modification loci. Overall, these results support the feasibility of low-pass nanopore sequencing as an exploratory framework for simultaneous cfDNA fragmentomic and epigenomic profiling in low-input studies.

DNA methylation, the covalent addition of methyl groups to cytosine (5mC) or adenine (6mA) in DNA, is a fundamental mechanism of epigenetic inheritance conserved from bacteria to humans. Fungi provide a uniquely informative window into the evolutionary logic of methylation systems. Spanning more than 1 billion years of diversification, the kingdom encompasses species that have lost cytosine methylation entirely, lineages that use 5mC to silence transposons and drive the irreversible genome-defense process known as repeat-induced point mutation (RIP), and early-diverging lineages, in which 6mA has emerged as a prominent chromatin mark. The methyltransferases underlying these strategies (DIM-2, RID, DNMT1-RFD, DNMT5, and the MT-A70 complex) and the recently characterized demethylases Dmt1 and CcTet are structurally and mechanistically distinct from their mammalian counterparts. Here we review the mechanisms, targets, and biological functions of fungal DNA methyltransferases and demethylases, incorporating cryo-EM structural insights into DIM-2 and DNMT5 catalysis, analyses of DNMT gene loss as a continuous evolutionary process, the antiviral role of DIM-2 in vegetative hyphae, and the emerging model of 6mA as a heritable regulatory mark in early-diverging lineages. By integrating these advances, this review offers the updated and comprehensive account of DNA methylation across fungi.

Background: Ogataea parapolymorpha DL-1 is a versatile thermotolerant organism with numerous applications in biotechnology, particularly in the production of recombinant proteins and the study of methanol metabolism and peroxisome functions. This study presents a comprehensive genome and methylome analysis of Ogataea parapolymorpha DL-1 using long-read sequencing technology. The research builds upon previous short-read sequencing efforts, revealing enhancements in genome assembly and epigenomic insights.Methods: We used long-read sequencing technology to achieve a telomere-to-telomere (T2T) genome assembly of Ogataea parapolymorpha DL-1. High-quality reads were obtained and assembled de novo, followed by polishing to enhance accuracy. The genome was analyzed to identify coding genes, telomeric motifs, rRNA genes, and methylation patterns, including the detection of 5mC and 6 mA modifications. Epigenetic features were further assessed and validated through liquid chromatography-mass spectrometry.Results: Key findings include the absence of 5 mC DNA modification and the presence of 6 mA in the genome, unusual telomere regulation mechanism based on the addition of non-telomeric dT and the introduction of long-read enhanced telomere-to-telomere assembly.Conclusion: This work provides deeper insights into the yeast’s genome organization and methylation patterns, contributing to the understanding of its genetics and therefore potential biotechnological applications.