Identification of fetal unmodified and 5-hydroxymethylated CG sites in maternal cell-free DNA for non-invasive prenatal testing

BackgroundMassively parallel sequencing of maternal cell-free DNA (cfDNA) is widely used to test fetal genetic abnormalities in non-invasive prenatal testing (NIPT). However, sequencing-based approaches are still of high cost. Building upon previous knowledge that placenta, the main source of fetal circulating DNA, is hypomethylated in comparison to maternal tissue counterparts of cfDNA, we propose that targeting either unmodified or 5-hydroxymethylated CG sites specifically enriches fetal genetic material and reduces numbers of required analytical sequencing reads thereby decreasing cost of a test.MethodsWe employed uTOPseq and hmTOP-seq approaches which combine covalent derivatization of unmodified or hydroxymethylated CG sites, respectively, with next generation sequencing, or quantitative real-time PCR.ResultsWe detected increased 5-hydroxymethylcytosine (5hmC) levels in fetal chorionic villi (CV) tissue samples as compared with peripheral blood. Using our previously developed uTOP-seq and hmTOP-seq approaches we obtained whole-genome uCG and 5hmCG maps of 10 CV tissue and 38 cfDNA samples in total. Our results indicated that, in contrast to conventional whole genome sequencing, such epigenomic analysis highly specifically enriches fetal DNA fragments from maternal cfDNA. While both our approaches yielded 100% accuracy in detecting Down syndrome in fetuses, hmTOP-seq maintained such accuracy at ultra-low sequencing depths using only one million reads. We identified 2164 and 1589 placenta-specific differentially modified and 5-hydroxymethylated regions, respectively, in chromosome 21, as well as 3490 and 2002 Down syndrome-specific differentially modified and 5-hydroxymethylated regions, respectively, that can be used as biomarkers for identification of Down syndrome or other epigenetic diseases of a fetus.ConclusionsuTOP-seq and hmTOP-seq approaches provide a cost-efficient and sensitive epigenetic analysis of fetal abnormalities in maternal cfDNA. The results demonstrated that T21 fetuses contain a perturbed epigenome and also indicated that fetal cfDNA might originate from fetal tissues other than placental chorionic villi. Robust covalent derivatization followed by targeted analysis of fetal DNA by sequencing or qPCR presents an attractive strategy that could help achieve superior sensitivity and specificity in prenatal diagnostics.Peer reviewe

A bisulfite-free approach for base-resolution analysis of genomic 5-carboxylcytosine

Due to an extreme rarity of 5-carboxylcytosine (5caC) in the mammalian genome, investigation of its role brings a considerable challenge. Methods based on bisulfite sequencing have been proposed for genome-wide 5caC analysis. However, bisulfite-based sequencing of scarcely abundant 5caC demands significant experimental and computational resources, increasing sequencing cost. Here, we present a bisulfite-free approach, caCLEAR, for high-resolution mapping of 5caCGs. The method uses an atypical activity of the methyltransferase eM.SssI to remove a carboxyl group from 5caC, generating unmodified CGs, which are localized by uTOP-seq sequencing. Validation of caCLEAR on model DNA systems and mouse ESCs supports the suitability of caCLEAR for analysis of 5caCGs. The 5caCG profiles of naive and primed pluripotent ESCs reflect their distinct demethylation dynamics and demonstrate an association of 5caC with gene expression. Generally, we demonstrate that caCLEAR is a robust economical approach that could help provide deeper insights into biological roles of 5caC

Precise genomic mapping of 5-hydroxymethylcytosine via covalent tether-directed sequencing

5-hydroxymethylcytosine (5hmC) is the most prevalent intermediate on the oxidative DNA demethylation pathway and is implicated in regulation of embryogenesis, neurological processes, and cancerogenesis. Profiling of this relatively scarce genomic modification in clinical samples requires cost-effective high-resolution techniques that avoid harsh chemical treatment. Here, we present a bisulfite-free approach for 5hmC profiling at single-nucleotide resolution, named hmTOP-seq (5hmC-specific tethered oligonucleotide-primed sequencing), which is based on direct sequence readout primed at covalently labeled 5hmC sites from an in situ tethered DNA oligonucleotide. Examination of distinct conjugation chemistries suggested a structural model for the tether-directed nonhomologous polymerase priming enabling theoretical evaluation of suitable tethers at the design stage. The hmTOP-seq procedure was optimized and validated on a small model genome and mouse embryonic stem cells, which allowed construction of single-nucleotide 5hmC maps reflecting subtle differences in strand-specific CG hydroxymethylation. Collectively, hmTOP-seq provides a new valuable tool for cost-effective and precise identification of 5hmC in characterizing its biological role and epigenetic changes associated with human disease

Multiomics analysis of neuroblastoma cells reveals a diversity of malignant transformations

Neuroblastoma (NB) is a pediatric cancer of the developing sympathetic nervous system that exhibits significant variation in the stage of differentiation and cell composition of tumors. Global loss of DNA methylation and genomic 5-hydroxymethylcytosine (5hmC) is a hallmark of human cancers. Here, we used our recently developed single-base resolution approaches, hmTOP-seq and uTOP-seq, for construction of 5hmC maps and identification of large partially methylated domains (PMDs) in different NB cell subpopulations. The 5hmC profiles revealed distinct signatures characteristic to different cell lineages and stages of malignant transformation of NB cells in a conventional and oxygen-depleted environment, which often occurs in tumors. The analysis of the cell-type-specific PMD distribution highlighted differences in global genome organization among NB cells that were ascribed to the same lineage identity by transcriptomic networks. Collectively, we demonstrated a high informativeness of the integrative epigenomic and transcriptomic research and large-scale genome structure in investigating the mechanisms that regulate cell identities and developmental stages of NB cells. Such multiomics analysis, as compared with mutational studies, open new ways for identification of novel disease-associated features which bring prognostic and therapeutic value in treating this aggressive pediatric disease

Distribution and regulatory roles of oxidized 5-methylcytosines in DNA and RNA of the basidiomycete fungi Laccaria bicolor and Coprinopsis cinerea

The formation of three oxidative DNA 5-methylcytosine (5mC) modifications (oxi-mCs)-5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC)-by the TET/JBP family of dioxygenases prompted intensive studies of their functional roles in mammalian cells. However, the functional interplay of these less abundant modified nucleotides in other eukaryotic lineages remains poorly understood. We carried out a systematic study of the content and distribution of oxi-mCs in the DNA and RNA of the basidiomycetes Laccaria bicolor and Coprinopsis cinerea, which are established models to study DNA methylation and developmental and symbiotic processes. Quantitative liquid chromatography-tandem mass spectrometry revealed persistent but uneven occurrences of 5hmC, 5fC and 5caC in the DNA and RNA of the two organisms, which could be upregulated by vitamin C. 5caC in RNA (5carC) was predominantly found in non-ribosomal RNA, which potentially includes non-coding, messenger and small RNA species. Genome-wide mapping of 5hmC and 5fC using the single CG analysis techniques hmTOP-seq and foTOP-seq pointed at involvement of oxi-mCs in the regulation of gene expression and silencing of transposable elements. The implicated diverse roles of 5mC and oxi-mCs in the two fungi highlight the epigenetic importance of the latter modifications, which are often neglected in standard whole-genome bisulfite analyses