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A Review of Next-Generation Sequencing Methods for Studying Epigenetics—Part 2



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  • A Review of Next-Generation Sequencing Methods for Studying Epigenetics—Part 2

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    In the first segment of our review of epigenetic sequencing methods, we covered methylation detection, chromatin accessibility, and protein-DNA interactions. This final part of our review will break outside of the standard analysis and cover some influential multidimensional methods.

    Spatial Epigenomics
    “Epigenetic assays, whether conducted at the single-cell or bulk level, have played a crucial role in unraveling the underlying mechanisms of diseases and the functions of various tissues,” said Colin Ng, Vice President of AtlasXomics. “However, there is an increasing need to understand these processes within their spatial context.” When combined with traditional sequencing methods, spatial technologies have the power to reveal another important layer of epigenetics.

    Spatial ATAC-Seq
    As highlighted by Deng et al. (2022), incorporating a spatial dimension into ATAC-Seq can be used to drastically improve our comprehension of epigenetics. “Our spatial ATAC assay empowers unbiased, genome-wide mapping of chromatin accessibility, providing valuable insights into DNA organization and regulation,” described Ng. “By identifying regions of open chromatin associated with gene expression, regulatory elements, and transcription factor binding sites, it enables the discovery of distinct cell types and their spatial relationships within the tissue context.”

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    Figure 1. An overview of the DBiT-seq procedure for spatial ATAC-seq (courtesy of AtlasXomics).

    AtlasXomics uses their platform, the DBiT-seq (Deterministic Barcoding in Tissue for spatial-omics sequencing), which pairs microfluidics with sequencing to perform their comprehensive epigenomic analysis. This spatial ATAC method is similar to the standard ATAC-Seq, in that a Tn5 transposase is used to fragment DNA at open chromatin regions and add on specific adapters. However, the samples are also applied to microfluidics, and barcodes are added onto the adapters that provide x- and y-spatial coordinates. After sequencing, the open regions of chromatin can be identified during data analysis and the barcodes can then be used to create spatial maps.

    Although this is a newer technology, there have still been some additional developments. Within the last year, academic and commercial groups have introduced creative solutions to improve the use of ATAC-Seq on FFPE tissue, explained Ng. “These solutions range from modifications in the tissue processing steps to changes in the downstream library preparation for sequencing. We look forward to adapting these solutions for our spatial platform so that we can help researchers profile the gold mine of information stored within their biobanked samples.”

    Spatial CUT&Tag
    “Unlike our spatial ATAC-Seq assay, CUT&Tag offers the advantage of selectively targeting specific histone modifications using antibodies,” said Ng. “This capability facilitates the examination of chromatin regions associated with these specific histone marks and their contribution to gene regulation. By profiling chromatin accessibility in proximity to these targeted proteins, CUT&Tag provides valuable insights into protein-DNA interactions, transcription factor binding, and the epigenetic modifications linked to the targeted proteins. This can provide a more granular understanding of epigenetic biomarkers of disease and histone modifications controlling chromatin structure.”

    The spatial CUT&Tag builds upon the methodology briefly explained in the first article, but in addition, it utilizes microfluidic deterministic barcoding followed by next-generation sequencing, similar to the spatial ATAC-Seq assay. This additional layer significantly increases the resolution to which researchers can investigate epigenetics. As highlighted by Ng, “With our spatial ATAC-Seq and CUT&Tag assays, we can leverage the comprehensive nature of NGS to profile the important yet subtle differences in chromatin and histone modifications directly in tissue, which we now believe play a major role in controlling disease pathogenesis and aging.”

    Ng also stated that there were several exciting new advancements used to better understand the complexities of gene regulation, such as “multi-modal CUT&Tag, the improved utility of CUT&Tag for a wider range of targets (e.g., low expressed targets, transcription factors), FFPE compatibility, and multiomics (epigenome + transcriptome/proteome).”

    3D Genomics
    “3D genomics involves exploring the 3-dimensional organization of DNA in the nucleus to reveal insights into the genome’s sequence, structure, and regulatory landscape,” said Ibrahim Jivanjee, Director of Product Management and Marketing at Arima Genomics. “Although techniques to understand the 3D organization of chromosomes were first described in the rat prolactin gene by Cullen et al. (1993) and then in yeast by Dekker et al. (2002), the field of 3D genomics is relatively nascent.”

    Jivanjee also highlighted the many levels of chromatin organization in the nucleus of cells, including chromosomal territories, compartments, domains, and looping structures that play a crucial role in coordinating regulatory interactions. Exploring these organizational levels can allow researchers to better understand gene regulation, silencing, and associated biological interactions.

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    Figure 2. The 3D genome is organized at multiple levels into chromosome territories, compartments, domains including topologically associated domains (TADs), and looping structures (courtesy of Arima Genomics).

    A popular and effective way of studying the 3D genome is through the use of HiC, a method that utilizes proximity ligation. “These solutions can help researchers understand how spatial and temporal changes in chromatin conformation alter gene regulation and cellular function,” stated Jivanjee. The HiC protocols work for a wide range of sample types and begin with cross-linking the samples. Then they are digested with restriction enzymes, labeled with biotin, and the DNA segments are ligated together. Along with some enrichment steps, a library prep is completed and the sample is sequenced and subsequently analyzed to generate a 3D genome map.

    There are several common choices for performing HiC. The first, Genome-Wide HiC, was described by Jivanjee as the most versatile solution. “This kit can help a researcher understand how the 3-dimensional organization of DNA in the nucleus can impact gene regulation, cellular development, and disease processes.” The next option, Capture HiC, is a more targeted approach that helps reduce sequencing costs and increase computational efficiency by limiting the data produced. Jivanjee explained that Arima offers a Promoter Capture HiC for characterizing promoter-enhancer interactions and a Custom Capture HiC that allows researchers to target regions of interest.

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    Figure 3. A brief overview of the HiC process (courtesy of Arima Genomics)

    “These solutions can help researchers understandhow spatial and temporal changes in chromatin conformation alter gene regulation and cellular function,” explained Jivanjee. “This information can be useful to understand spatial relationships and how they impact gene regulation, understand cellular development, study disease processes, understand epigenetic mechanisms, and identify structural elements in a genome including compartments, TADs, and loops.”

    Another important method for investigating the 3D genome is HiChIP, a chromatin conformation capture technique that combines chromatin immunoprecipitation (ChIP) with high-throughput chromosome conformation capture (HiC). This method, Jivanjee stated, “offers a way to measure active gene regulatory interactions as part of multiomics analysis of transcriptional protein binding and chromatin interactions.”

    During the HiChIP process, the sample is still fixed through proximal ligation but is then sheared and bound to an antibody. Afterward, the sample is immunoprecipitated and subjected to quality control. The samples are then converted into sequencing libraries and the resulting data is analyzed to determine important regulatory interactions. “This solution can help a researcher discover active gene regulatory interactions and can be useful as part of multiomics analyses of transcriptional protein binding and chromatin interactions,” said Jivanjee.

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    Figure 4. An overview of the HiChIP process (courtesy of Arima Genomics)

    One of the emerging trends with these 3D genomics methods is the widespread incorporation of multiomics. As explained by Jivanjee, “Transcriptional regulation is essential to the diverse function observed across cell type, time, disease states, the environment, genetic mutations, and many more factors. To explore how any one of these variables impacts gene expression, scientists frequently use transcriptomics to detect differential expression of genes. This compelling technique has significantly added to our understanding of transcriptional regulation. Still, RNA sequencing does not provide any information on what is causing that altered gene expression. And that is where 3D genomics can truly add value as part of a multiomics approach.”

    1. Deng Y, Marek Bartosovic, Kukanja P, et al. Spatial-CUT&Tag: Spatially resolved chromatin modification profiling at the cellular level. Science. 2022;375(6581):681-686. doi:https://doi.org/10.1126/science.abg7216
    2. Cullen KE, Kladde MP, Seyfred MA. Interaction Between Transcription Regulatory Regions of Prolactin Chromatin. Science. 1993;261(5118):203-206. doi:https://doi.org/10.1126/science.832789
    3. Dekker J, Rippe K, Dekker M, Kleckner N. Capturing Chromosome Conformation. Science. 2002;295(5558):1306-1311. doi:https://doi.org/10.1126/science.1067799
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