Search

5-methylcytosine and 5-hydroxymethylcytosine are epigenetic modifications involved in gene regulation and cancer. Here, we describe a new, simple, and high-throughput platform for multi-colour epigenetic analysis. The novelty of our approach is the ability to multiplex methylation and de-methylation signals in the same assay. We utilize an engineered methyltransferase enzyme that recognizes and labels all unmodified CpG sites with a fluorescent cofactor. In combination with the already established labelling of the de-methylation mark 5-hydroxymethylcytosine via enzymatic glycosylation, we obtained a robust platform for simultaneous epigenetic analysis of these marks. We assessed the global epigenetic levels in multiple samples of colorectal cancer and observed a reduction in 5-hydroxymethylcytosine levels, but no change in DNA methylation levels between sick and healthy individuals. We also measured epigenetic modifications in chronic lymphocytic leukaemia and observed a decrease in both modification levels. Our results indicate that this assay may be used for the epigenetic characterization of clinical samples for research and patient management.

Cancer cells display complex genomic aberrations that include large-scale genetic rearrangements and epigenetic modulation that are not easily characterized by short-read sequencing. We present a method for simultaneous profiling of long-range genetic/epigenetic changes in matched cancer samples. Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Most ccRCC cases demonstrate somatic genomic alterations involving the short arm of chromosome 3 (3p), most often targeting the von Hippel–Lindau (VHL) gene. Aiming to identify somatic alterations that characterize early stage ccRCC, we performed comprehensive genetic, cytogenetic and epigenetic analyses comparing ccRCC tumor to adjacent non-tumorous tissue. Optical genome mapping identified genomic aberrations such as structural and copy number variations, complementing exome-sequencing results. Single-molecule methylome and hydroxymethylome mapping revealed multiple differential regions, some of them known to be associated with ccRCC pathogenesis. Among them, metabolic pathways were significantly enriched. Moreover, significant global epigenetic differences were detected between the tumor and the adjacent non-tumorous tissue, and a correlation between epigenetic signals and gene expression was found. This is the first reported comparison of a human tumor and a matched tissue by optical genome/epigenome mapping, revealing well-established and novel somatic aberrations.

Epigenetic transformations may provide early indicators for cancer and other disease. Specifically, the amount of genomic 5-hydroxymethylcytosine (5-hmC) was shown to be globally reduced in a wide range of cancers. The integration of this global biomarker into diagnostic workflows is hampered by the limitations of current 5-hmC quantification methods. Here we present and validate a fluorescence-based platform for high-throughput and cost-effective quantification of global genomic 5-hmC levels. We utilized the assay to characterize cancerous tissues based on their 5-hmC content, and observed a pronounced reduction in 5-hmC level in various cancer types. We present data for glioblastoma, colorectal cancer, multiple myeloma, chronic lymphocytic leukemia and pancreatic cancer, compared to corresponding controls. Potentially, the technique could also be used to follow response to treatment for personalized treatment selection. We present initial proof-of-concept data for treatment of familial adenomatous polyposis.

Mapping DNA damage and its repair has immense potential in understanding environmental exposures, their genotoxicity, and their impact on human health. Monitoring changes in genomic stability also aids in the diagnosis of numerous DNA-related diseases, like cancer, and assists in monitoring their progression and prognosis. However, genome-wide maps of DNA damage distribution are challenging to produce. Here we describe the localization of DNA damage and repair loci by Repair Assisted Damage Detection sequencing – RADD-Seq. Based on the enrichment of damage lesions coupled with a pull-down assay and followed by next generation sequencing, this method is easy to perform and can produce compelling results with minimal coverage. RADD-seq enables the localization of both DNA damage and repair sites for a wide range of single-strand damage types. Using this technique, we created a genome-wide map of oxidative DNA damage loci before and after repair. Oxidative lesions were heterogeneously distributed along the human genome, with less damage occurring in tight chromatin regions. Furthermore, we showed repair is prioritized for highly expressed, essential genes and in open chromatin regions. RADD-seq sheds light on cellular repair mechanisms and capable of identifying genomic hotspots prone to mutation.

MotivationWhile promoter methylation is associated with reinforcing fundamental tissue identities, the methylation status of distant enhancers was shown by genome-wide association studies to be a powerful determinant of cell-state and cancer. With recent availability of long-reads that report on the methylation status of enhancer-promoter pairs on the same molecule, we hypothesized that probing these pairs on the single-molecule level may serve the basis for detection of rare cancerous transformations in a given cell population. We explore various analysis approaches for deconvolving cell-type mixtures based on their genome-wide enhancer-promoter methylation profiles.ResultsTo evaluate our hypothesis we examine long-read optical methylome data for the GM12787 cell line and myoblast cell lines from two donors. We identified over 100,000 enhancer-promoter pairs that co-exist on at least 30 individual DNA molecules per pair. We developed a detailed methodology for mixture deconvolution and applied it to estimate the proportional cell compositions in synthetic mixtures based on analyzing their enhancer-promoter pairwise methylation. We found our methodology to lead to very accurate estimates, outperforming our promoter-based deconvolutions. Moreover, we show that it can be generalized from deconvolving different cell types to subtle scenarios where one wishes to deconvolve different cell populations of the same cell-type.AvailabilityThe code used in this work to analyze single-molecule Bionano Genomics optical maps is available via the GitHub repositoryhttps://github.com/ebensteinLab/Single_molecule_methylation_in_EP.Contactuv@post.tau.ac.il(Y.E),roded@tauex.tau.ac.il(R.S)

Knowing the amount and type of DNA damage is of great significance for a broad range of clinical and research applications. However, existing methods are either lacking in their ability to distinguish between types of DNA damage or limited in their sensitivity and reproducibility. The method described herein enables rapid and robust quantification of type-specific single-strand DNA damage. The method is based on repair-assisted damage detection (RADD) by which fluorescent nucleotides are incorporated into DNA damage sites using type-specific repair enzymes. Up to 90 DNA samples are then deposited on a multiwell glass slide, and analyzed by a conventional slide scanner for quantification of DNA damage levels. Accurate and sensitive measurements of oxidative or UV-induced DNA damage levels and repair kinetics are presented for both in vitro and in vivo models.

The human genome contains multiple layers of information that extend beyond the genetic sequence. In fact, identical genetics do not necessarily yield identical phenotypes as evident for the case of two different cell types in the human body. The great variation in structure and function displayed by cells with identical genetic background is attributed to additional genomic information content. This includes large-scale genetic aberrations, as well as diverse epigenetic patterns that are crucial for regulating specific cell functions. These genetic and epigenetic patterns operate in concert in order to maintain specific cellular functions in health and disease. Single-molecule optical genome mapping is a high-throughput genome analysis method that is based on imaging long chromosomal fragments stretched in nanochannel arrays. The access to long DNA molecules coupled with fluorescent tagging of various genomic information presents a unique opportunity to study genetic and epigenetic patterns in the genome at a single-molecule level over large genomic distances. Optical mapping entwines synergistically chemical, physical, and computational advancements, to uncover invaluable biological insights, inaccessible by sequencing technologies. Here we describe the method’s basic principles of operation, and review the various available mechanisms to fluorescently tag genomic information. We present some of the recent biological and clinical impact enabled by optical mapping and present recent approaches for increasing the method’s resolution and accuracy. Finally, we discuss how multiple layers of genomic information may be mapped simultaneously on the same DNA molecule, thus paving the way for characterizing multiple genomic observables on individual DNA molecules.