| Abstract [eng] |
5-hydroxymethylcytosine (5hmC) is an important DNA modification that plays a gene regulatory role in human physiological and pathological states. The so-called “gold standard” single-base resolution methods for 5hmC profiling are based on bisulfite treatment, which causes DNA loss and imposes challenges in data analysis due to altered base composition. Furthermore, the high cost of whole-genome sequencing makes these approaches prohibitive for the disease or population studies. This work describes the development of a novel cost-effective 5hmC profiling method based on the covalent DNA labeling – hmTOP-seq (5hmC-specific tethered oligonucleotide-primed sequencing). We demonstrated the main advantages of hmTOP-seq: its high resolution and high specificity for 5hmC, good reproducibility, high correlation with other methods, and ability to provide DNA strand-specific hydroxymethylation information. The developed hmTOP-seq method was successfully applied in the epigenomic studies of human diseases – neuroblastoma (NB) and trisomy of the 21st chromosome (Down syndrome). Using hmTOP-seq and uTOP-seq, we performed a detailed multi-omic (5hmC, unmodified CG and transcriptomic) analysis of different NB cell types, which allowed us to determine hypoxia-induced changes in gene hydroxymethylation and expression, as well as comprehensively investigate differences among various NB cells. We also demonstrated that hmTOP-seq method can be applied for the epigenetic non-invasive prenatal testing of the fetal trisomy of chromosome 21 from blood plasma cell-free DNA of pregnant women using quantitative PCR or DNA sequencing. |
