NGS

M.A. Abdurashitov, A.G. Akishev, S.Kh. Degtyarev SibEnzyme Ltd, Novosibirsk Corresponding author: M.A. Abdurashitov, SibEnzyme Ltd., 2/12 Ak. Timakova Street,Novosibirsk 630117, Russia; Tel.: +7 (383) 333-4991; Fax: +7 (383) 333-6853; E-mail: abd@sibenzyme.ru Abstract The 5-methylcytosine-dependent DNA endonuclease GlaI recognizes and cleaves DNA sites with the sequence 5′-R(5mC)GY-3′ / 3′-YG(5mC)R-5′, which are formed de novo in the human genome by the DNA methyltransferase DNMT3. Owing to this specificity, GlaI is efficiently used to determine the methylation status of RCGY sites in the human genome. Genome-wide mapping of R(5mC)GY sites can be performed by next-generation sequencing (NGS) of DNA hydrolysates generated by GlaI digestion, whereas analysis of individual DNA fragments can be carried out using GlaI-PCR analysis. In this study, we compared the results of NGS analysis of GlaI DNA hydrolysates with data obtained by GlaI-PCR analysis of regulatory regions of 11 tumor suppressor genes in DNA from human cell lines L68 (normal lung fibroblasts), Raji (Burkitt lymphoma), and U937 (histiocytic lymphoma). It was shown that in L68 cells most of the analyzed regulatory regions are unmethylated, whereas methylation of these regions is observed in the malignant cell lines Raji and U937. With the exception of a single region, for all other analyzed fragments the methylation status determined by GlaI-PCR analysis fully correlates with the number of R(5mC)GY sites detected by NGS. The only discrepancy between the two methods was identified for the regulatory region of the RASSF1A gene in L68 cells and was attributed to the presence of allele-specific methylation, which was confirmed by additional real-time GlaI-PCR analysis. Taking allele-specific methylation of the RASSF1A regulatory region into account, complete concordance was observed between the data obtained by NGS analysis of GlaI DNA hydrolysates and GlaI-PCR analysis of regulatory regions of the studied tumor suppressor genes in the human cell lines L68, Raji, and U937. Thus, NGS analysis of GlaI DNA hydrolysates and GlaI-PCR analysis are complementary approaches that enable reliable identification of aberrantly methylated genomic regions in normal and malignant cells, making them promising tools for epigenetic research and DNA-based diagnostics. Keywords: 5-methylcytosine; DNA methylation; methyl-dependent restriction endonucleases;GlaI; NGS analysis; GlaI-PCR analysis; epigenetic DNA diagnostics; allele-specific methylation. DOI: 10.26213/3034-4298.2025.6.1.001 Citation: M.A. Abdurashitov, A.G. Akishev, S.Kh. Degtyarev (2025) Determination of the Methylation Status of RCGY Sites in the Human Genome: A Comparison of NGS Analysis of GlaI DNA Hydrolysates and GlaI-PCR Data, Epigenetic DNA diagnostics, vol 2025(1), DOI: 10.26213/3034-4298.2025.6.1.001 This work is available at Creative Commons…

DNA methylation in human genome is important for the cells specialization and functioning. An abnormal methylation of the regulation regions of some genes may cause the genes silencing and this phenomenon is often detected in cancer cells. Determination of differences of the genome-wide methylation in normal and tumor cells is useful for understanding the carcinogenesis process and for development of new methods of epigenetic diagnostics. The positions of methylated RCGY sites in the genomes of Raji, U-937 and L68 human cell lines have been determined using the previously developed method of massive parallel sequencing of Glal fragments. A comparison of the obtained data has revealed significant differences in methylation of CpG islands, putative regulatory regions and some repetitive DNA families between genomes of malignant and non-malignant cells. GO enrichment analysis of genes with highly methylated regulatory regions has shown the possible metabolic processes, which may be affected epigenetically in carcinogenesis. The new method allows to determine positions of many modified cytosine bases in the genomes and may be a simple alternative to the existing methods of genome-wide methylation analysis.