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 separated flow around a balance-mounted, 60-deg sweptback, semispan delta wing with a sharp leading edge was controlled using zero-mass-flux periodic excitation from a segmented leading-edge slot. Excitation was generated by cavity-installed piezoelectric actuators operating at resonance with amplitude modulation (AM) and burst mode (BM) signals being used to achieve reduced frequencies (scaled with the freestream velocity and the root chord) in the range from O(1) to O(10). Results of a parametric investigation, studying the effects of AM frequency, BM duty cycle and frequency, excitation amplitude, location of the actuation along the leading edge, and optimal phase difference between the actuators, as well as the Reynolds number, are reported and discussed. Balance data were supplemented by upper surface static pressure measurements and particle image velocimetry (PIV) data. Order unity reduced-frequency modulation of the high-frequency carrier wave increased the normal force generated by the delta wing most effectively. BM with a duty cycle that was as low as 5% was more effective than the amplitude-modulated signal with larger peak excitation velocity and an order of magnitude larger momentum input. PIV data suggest that excitation enhances the momentum transfer across the shear layer, downstream of the original vortex breakdown location, generating a streamwise vortex the size of which is commensurate with the local wing span. Nomenclatureleading-edge length D = drag force DCy = duty cycle, n f m / f r FM = figure of merit, η/(C L /C D ) baseline F + = nondimensional excitation frequency, ( f c/U ∞ ) f = excitation frequency; either f m or f r , depends on excitation type f m = modulating frequency f r = actuators resonance frequency K = number of active actuator elements L = length of separated region n = number of excitation cycles p = local pressure Q = in-plane velocity, √ (w 2 + v 2 ) q = freestream dynamic pressure, ρU 2 ∞ /2 Re = root chord Reynolds number, U ∞ c/ν T m = input signal modulation period T r = period of actuators' sine wave U p = slot exit peak velocity U ∞ = freestream velocity u f = fast Fourier transform amplitude results at f = f m u = rms of velocity fluctuations u s = u at the slot's exit V rms = rms excitation voltage v, w = velocity components of the flow in the y, z directions W= actuator input power, W X R = distance from actuator to reattachment area X TE = distance from actuator to trailing edge x, y, z = Cartesian coordinates, (Fig. 1) x , y = rotated coordinates, (Fig. 1) α = angle of attack α s = stall α η = aerodynamic efficiency, C L /(C D + C E ) η R = spanwise location of the center of pressure, origin at tunnel wall, C R /C N ν = kinematic viscosity ρ = air density = phase angle between input signals to each actuator χ M = streamwise location of the center of pressure, origin at midchord, C M /C N = dimensionless vorticity, (∂v/∂x − ∂u/∂ y)/U ∞ c

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.

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.

DNA methylation, specifically, methylation of cytosine (C) nucleotides at the 5-carbon position (5-mC), is the most studied and significant epigenetic modification. Here we developed a chemoenzymatic procedure to fluorescently label non-methylated cytosines in CpG context, allowing epigenetic profiling of single DNA molecules spanning hundreds of thousands of base pairs. We used a CpG methyltransferase with a synthetic S-adenosyl-l-methionine cofactor analog to transfer an azide to cytosines instead of the natural methyl group. A fluorophore was then clicked onto the DNA, reporting on the amount and position of non-methylated CpGs. We found that labeling efficiency was increased up to 2-fold by the addition of a nucleosidase, presumably by degrading the inactive by-product of the cofactor after labeling, preventing its inhibitory effect. We used the method to determine the decline in global DNA methylation in a chronic lymphocytic leukemia patient and then performed whole-genome methylation mapping of the model plant Arabidopsis thaliana. Our genome maps show high concordance with published bisulfite sequencing methylation maps. Although mapping resolution is limited by optical detection to 500–1000 bp, the labeled DNA molecules produced by this approach are hundreds of thousands of base pairs long, allowing access to long repetitive and structurally variable genomic regions.

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.

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.

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