Dam Assisted Fluorescent Tagging of Chromatin Accessibility (DAFCA) for Optical Genome Mapping in Nanochannel Arrays

Abstract

Proteins and enzymes in the cell nucleus require physical access to their DNA target sites in order to perform genomic tasks such as gene activation and transcription. Hence, chromatin accessibility is a central regulator of gene expression, and its genomic profile holds essential information on the cell type and state. We utilized the E. coli Dam methyltransferase in combination with a fluorescent cofactor analogue to generate fluorescent tags in accessible DNA regions within the cell nucleus. The accessible portions of the genome are then detected by single-molecule optical genome mapping in nanochannel arrays. This method allowed us to characterize long-range structural variations and their associated chromatin structure. We show the ability to create whole-genome, allele-specific chromatin accessibility maps composed of long DNA molecules extended in silicon nanochannels.

Long-Read Structural and Epigenetic Profiling of a Kidney Tumor-Matched Sample with Nanopore Sequencing and Optical Genome Mapping

“…Figure 5.E . shows an example of a large repetitive element containing a group of genes from the GAGE family, poorly represented in the hg38 reference (the entire array spans ∼190 kbp in the reference, with a gap within these coordinates) (Nifker et al 2023). Long molecules spanning the entire uncharacterized region in OGM aided in assembling a contig of the full repetitive element, and the 5hmC tags on these molecules provided the 5hmC profile along the unknown region.…”

“…The 5hmC profile generated by both methods and displayed in Figure 5.D reveals a broadly correlated profile, but with distinct amplitude variations between the different datasets, in line with the average global levels. Figure 5.E shows an example of a large repetitive element containing a group of genes from the GAGE family, poorly represented in the hg38 reference (the entire array spans ∼190 kbp in the reference, with a gap within these coordinates) 57 . Long molecules spanning the entire uncharacterized region in OGM aided in assembling a contig of the full repetitive element, and the 5hmC tags on these molecules provided the 5hmC profile along the unknown region.…”

OM2Seq: learning retrieval embeddings for optical genome mapping

“…Optical genome mapping (OGM) is a method for mapping optical images of linearly extended and labeled DNA fragments to reference genome sequences ( Neely et al 2010 , Michaeli and Ebenstein 2012 , Jeffet et al 2021 ). It has shown potential in a range of applications, including the detection of structural and copy-number variations ( Ebert et al 2021 ), identification of DNA damage ( Torchinsky et al 2019 ), epigenomic profiling ( Gabrieli et al 2018 , Sharim et al 2019 , Gabrieli et al 2022 , Nifker et al 2023 ), and microbial species identification ( Grunwald et al 2015 , Wand et al 2019 , Bouwens et al 2020 , Müller et al 2020 ). OGM’s strengths lie in its ability to image genome fragments up to megabase size and detect both large-scale structural and copy number variations, as well as working with low quantities of target DNA, which is particularly useful in cultivation-free pathogen identification ( Müller et al 2020 , Nyblom et al 2023 ).…”

Detecting Protein-DNA Binding in Single Molecules using Antibody Guided Methylation

“…This enzyme was also used to observe the regulatory architectures of chromatin fibers 7 . In a non-sequencing approach, DNA accessibility of single molecules was probed by enzymatic attachment of fluorescent cofactors, followed by optical genome mapping in nanochannel arrays 8 .…”