Motivation While 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. Results To 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. Availability The code used in this work to analyze single-molecule Bionano Genomics optical maps is available via the GitHub repository https://github.com/ebensteinLab/Single_molecule_methylation_in_EP . Contact uv@post.tau.ac.il (Y.E), roded@tauex.tau.ac.il (R.S)

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, such as cancer, and assists in monitoring their progression and prognosis. Developments in recent years have enabled unprecedented sensitivity in quantifying the global DNA damage dose in cells via fluorescence-based analysis down to the single-molecule level. 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 the oxidation DNA damage lesion 8-oxo-7,8-dihydroguanine before and after repair. Oxidation 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 is capable of identifying genomic hotspots prone to mutation.

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.

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.

DNA combing is a widely used method for stretching and immobilising DNA molecules on a surface. Fluorescent labelling of genomic information enables high-resolution optical analysis of DNA at the single-molecule level. Despite its simplicity, the application of DNA combing in diagnostic workflows is still limited, mainly due to difficulties in analysing multiple small-volume DNA samples in parallel. Here, we report a simple and versatile microfluidic DNA combing technology (μDC), which allows manipulating, stretching and imaging of multiple, microliter scale DNA samples by employing a manifold of parallel microfluidic channels. Using DNA molecules with repetitive units as molecular rulers, we demonstrate that the μDC technology allows uniform stretching of DNA molecules. The stretching ratio remains consistent along individual molecules as well as between different molecules in the various channels, allowing simultaneous quantitative analysis of different samples loaded into parallel channels. Furthermore, we demonstrate the application of μDC to characterise UVB-induced DNA damage levels in human embryonic kidney cells and the spatial correlation between DNA damage sites. Our results point out the potential application of μDC for quantitative and comparative single-molecule studies of genomic features. The extremely simple design of μDC makes it suitable for integration into other microfluidic platforms to facilitate high-throughput DNA analysis in biological research and medical point-of-care applications.

We report on the development of a methylation analysis workflow for optical detection of fluorescent methylation profiles along chromosomal DNA molecules. In combination with Bionano Genomics genome mapping technology, these profiles provide a hybrid genetic/epigenetic genome-wide map composed of DNA molecules spanning hundreds of kilobase pairs. The method provides kilobase pair–scale genomic methylation patterns comparable to whole-genome bisulfite sequencing (WGBS) along genes and regulatory elements. These long single-molecule reads allow for methylation variation calling and analysis of large structural aberrations such as pathogenic macrosatellite arrays not accessible to single-cell second-generation sequencing. The method is applied here to study facioscapulohumeral muscular dystrophy (FSHD), simultaneously recording the haplotype, copy number, and methylation status of the disease-associated, highly repetitive locus on Chromosome 4q.

The epigenetic mark 5-hydroxymethylcytosine (5-hmC) is a distinct product of active enzymatic demethylation that is linked to gene regulation, development and disease. Genome-wide 5-hmC profiles generated by short-read next-generation sequencing are limited in providing long-range epigenetic information relevant to highly variable genomic regions, such as the 3.7 Mbp disease-related Human Leukocyte Antigen (HLA) region. We present a long-read, single-molecule mapping technology that generates hybrid genetic/epigenetic profiles of native chromosomal DNA. The genome-wide distribution of 5- hmC in human peripheral blood cells correlates well with 5-hmC DNA immunoprecipitation (hMeDIP) sequencing. However, the long read length of 100 kbp-1Mbp produces 5-hmC profiles across variable genomic regions that failed to showup in the sequencing data. In addition, optical 5-hmC mapping shows strong correlation between the 5-hmC density in gene bodies and the corresponding level of gene expression. The single molecule concept provides information on the distribution and coexistence of 5-hmC signals at multiple genomic loci on the same genomic DNA molecule, revealing long-range correlations and cell-to-cell epigenetic variation.

Next generation sequencing (NGS) is challenged by structural and copy number variations larger than the typical read length of several hundred bases. Third-generation sequencing platforms such as single-molecule real-time (SMRT) and nanopore sequencing provide longer reads and are able to characterize variations that are undetected in NGS data. Nevertheless, these technologies suffer from inherent low throughput which prohibits deep sequencing at reasonable cost without target enrichment. Here, we optimized Cas9-Assisted Targeting of CHromosome segments (CATCH) for nanopore sequencing of the breast cancer gene BRCA1. A 200 kb target containing the 80 kb BRCA1 gene body and its flanking regions was isolated intact from primary human peripheral blood cells, allowing long-range amplification and long-read nanopore sequencing. The target was enriched 237-fold and sequenced at up to 70× coverage on a single flow-cell. Overall performance and single-nucleotide polymorphism (SNP) calling were directly compared to Illumina sequencing of the same enriched sample, highlighting the benefits of CATCH for targeted sequencing. The CATCH enrichment scheme only requires knowledge of the target flanking sequence for Cas9 cleavage while providing contiguous data across both coding and non-coding sequence and holds promise for characterization of complex disease-related or highly variable genomic regions.

The epigenetic mark 5-hydroxymethylcytosine (5-hmC) is a distinct product of active DNA demethylation that is linked to gene regulation, development, and disease. In particular, 5-hmC levels dramatically decline in many cancers, potentially serving as an epigenetic biomarker. The noise associated with next-generation 5-hmC sequencing hinders reliable analysis of low 5-hmC containing tissues such as blood and malignant tumors. Additionally, genome-wide 5-hmC profiles generated by short-read sequencing are limited in providing long-range epigenetic information relevant to highly variable genomic regions, such as the 3.7 Mbp disease-related Human Leukocyte Antigen (HLA) region. We present a long-read, highly sensitive single-molecule mapping technology that generates hybrid genetic/epigenetic profiles of native chromosomal DNA. The genome-wide distribution of 5-hmC in human peripheral blood cells correlates well with 5-hmC DNA immunoprecipitation (hMeDIP) sequencing. However, the long single-molecule read-length of 100 kbp to 1 Mbp produces 5-hmC profiles across variable genomic regions that failed to show up in the sequencing data. In addition, optical 5-hmC mapping shows a strong correlation between the 5-hmC density in gene bodies and the corresponding level of gene expression. The single-molecule concept provides information on the distribution and coexistence of 5-hmC signals at multiple genomic loci on the same genomic DNA molecule, revealing long-range correlations and cell-to-cell epigenetic variation.

Reduced representation methylation profiling is a method of analysis in which a subset of CpGs is used to report the overall methylation status of the probed genomic regions. This approach has been widely adopted for genome-scale bisulfite sequencing since it requires fewer sequencing reads and uses significantly less starting material than whole-genome analysis. Consequently, this method is suitable for profiling medical samples and single cells at high throughput and reduced costs. Here, we use this concept in order to create a pattern of fluorescent optical methylation profiles along individual DNA molecules. Reduced representation optical methylation mapping (R2OM2) in combination with Bionano Genomics next generation genome mapping (NGM) technology provides a hybrid genetic/epigenetic genome map of individual chromosome segments spanning hundreds of kilobase pairs (kbp). These long reads, along with the single-molecule resolution, allow for epigenetic variation calling and methylation analysis of large structural aberrations such as pathogenic macrosatellite arrays not accessible to single-cell next generation sequencing (NGS). We apply this method to facioscapulohumeral dystrophy (FSHD) showing both structural variation and hypomethylation status of a disease-associated, highly repetitive locus on chromosome 4q.

ABSTRACT Variations in the genetic code, from single point mutations to large structural or copy number alterations, influence susceptibility, onset, and progression of genetic diseases and tumor transformation. Next-generation sequencing analysis is unable to reliably capture aberrations larger than the typical sequencing read length of several hundred bases. Long-read, single-molecule sequencing methods such as SMRT and nanopore sequencing can address larger variations, but require costly whole genome analysis. Here we describe a method for isolation and enrichment of a large genomic region of interest for targeted analysis based on Cas9 excision of two sites flanking the target region and isolation of the excised DNA segment by pulsed field gel electrophoresis. The isolated target remains intact and is ideally suited for optical genome mapping and long-read sequencing at high coverage. In addition, analysis is performed directly on native genomic DNA that retains genetic and epigenetic composition without amplification bias. This method enables detection of mutations and structural variants as well as detailed analysis by generation of hybrid scaffolds composed of optical maps and sequencing data at a fraction of the cost of whole genome sequencing.

T4 β-glucosyltransferase is used to tag 5hmC sites with a fluorescent reporter molecule. The enzyme is fed with a synthetic cofactor UDP-6-N3-Glu, resulting in covalent attachment of a functional azide at the 5hmC site. This azide is further reacted with a DBCO functionalized fluorophore via a copper-free "click" chemistry reaction to generate the fluorescently labeled 5hmC. enzymatic fluorescent Chemo hmC 5 labeling of nm wide 45 DNA is stretched in silicon nanochannels

DNA damage and repair are linked to fundamental biological processes such as metabolism, disease, and aging. Single-strand lesions are the most abundant form of DNA damage; however, methods for characterizing these damage lesions are lacking. To avoid double-strand breaks and genomic instability, DNA damage is constantly repaired by efficient enzymatic machinery. We take advantage of this natural process and harness the repair capacity of a bacterial enzymatic cocktail to repair damaged DNA in vitro and incorporate fluorescent nucleotides into damage sites as part of the repair process. We use single-molecule imaging to detect individual damage sites in genomic DNA samples. When the labeled DNA is extended on a microscope slide, damage sites are visualized as fluorescent spots along the DNA contour, and the extent of damage is easily quantified. We demonstrate the ability to quantitatively follow the damage dose response to different damaging agents as well as repair dynamics in response to UV irradiation in several cell types. Finally, we show the modularity of this single-molecule approach by labeling DNA damage in conjunction with 5-hydroxymethylcytosine in genomic DNA extracted from mouse brain tissue.