Long Read Sequencing: A Game Changer in Mapping Complex Epigenetic Landscapes

Epigenetic modifications, which consist of chemical modifications of DNA and histone proteins, play a pivotal role in regulating gene expression without altering the underlying DNA sequence. Similar to the advancements in genomics field, epigenomics has been revolutionized by the advent of long-read sequencing (LRS) technologies. Unlike traditional short-read sequencing (SRS) methods, which often fail to capture the full complexity of the epigenome, LRS enables the direct detection of DNA modifications by sequencing long DNA fragments, thus providing a more detailed and comprehensive view of the epigenome. This technology facilitates the investigation of complex chromatin landscapes with high resolution, providing opportunities for multi-omics studies that combine epigenomics and transcriptomics data.

Advantages of Long-Read Sequencing for Epigenomics

LRS offers several advantages over SRS methods due to it ability to generate substantially longer reads of DNA molecules. One of the most significant benefits is the direct detection of comprehensive epigenetic profile without additional processing steps. Its PCR-free nature  leads to more accurate quantification of epigenetic modifications by reducing PCR-induced biases. Additionally, LRS facilitates haplotype-resolved epigenomics by spanning entire haplotype blocks and enables us to distinguish between maternal and paternal alleles. Finally, LRS provides superior coverage of highly repetitive regions (HRRs) and duplications, which are often difficult to analyze with SRS, thereby allowing for more accurate mapping of challenging genomic regions (1).

Major Applications of Long-Read Sequencing in Epigenomics Field

LRS technologies have enabled the development of novel assays for studying various chromatin features. Major applications include direct DNA methylation detection, chromatin accessibility profiling, direct mapping of protein-DNA interactions and 3D genome organization study.

• Direct methylation detection. Detecting DNA methylation with SRS requires specialized methods like bisulfite conversion, affinity enrichment, and endonuclease cleavage, which can introduce biases. Bisulfite conversion may cause DNA degradation and incomplete conversion, while affinity enrichment may bias toward CpG-rich regions.  Moreover, these methods can only capturing 5-methylcytosine. In contrast, LRS enables researchers to explore the full diversity and complexity of DNA modifications, including 5-hydroxymethylcytosine (5hmC), N6-methyladenine (6mA), and N4-methylcytosine (4mC), without requiring separate library preparation (1).
• Chromatin accessibility profiling. Limitations of SRS-based approaches make it challenging to detect complex cis-acting chromatin states. Moreover, SRS approaches fail to resolve chromatin accessibility at segmental duplications and high repetitive regions. Unlike SRS methods, LRS enables researchers to explore open chromatin regions with high resolution, providing insight into nucleosome positioning and regulatory element accessibility.
• Direct mapping of protein–DNA interactions. Over the past decades, SRS-based methods such as bulk CHIP-seq have significantly enhanced our comprehension of protein-DNA interactions in gene regulation mechanisms. However, these methods have limitations, such as their inability to measure multiple protein interactions with the same DNA molecule (2). Unlike SRS technologies, LRS-based methods, including DiMeLo-seq and nanoHiMe-seq (3), facilitate the precise identification of transcription factor binding sites and histone modifications, thereby providing a more detailed understanding of regulatory mechanisms at the single-molecule level.
• 3D genome organization study. Eukaryotic genomes are organized in a complex and multiscale three-dimensional architecture. Studying this can provide deeper mechanistic insights into nuclear organization, function, and gene regulation. LRS-based protocols such as Pore-C (4) and HiPore-C provide insights into higher-order chromatin interactions and multiway contacts between genomic loci, allowing researchers to study 3D genome organization with an unprecedented level of detail  (5).
• LRS methods for single-cell DNA methylome and chromatin accessibility analysis. Emerging LRS methods use long reads to detect epigenetic features of full-length CpG islands and gene promoters. This allows for profiling allele-specific epigenetic states within individual cells, including DNA methylation and chromatin accessibility of imprinting control regions (6).

The Future of Multi-Omics Research

By integrating LRS-based epigenomics data with long-read transcriptomics simultaneously, researchers can uncover novel insights into gene regulation and chromatin dynamics. Future developments in LRS will likely focus on simultaneous multi-modal sequencing, which aims to capture DNA methylation, chromatin accessibility, and transcriptomic data within the same biological sample (1), providing a more holistic view of gene regulation (1,7). Additionally, the field will benefit from improved computational tools. Advanced machine-learning algorithms and statistical models will enhance the interpretation of LRS datasets, enabling more accurate detection of epigenetic modifications. Additionally, the advancement of LRS-based multi-omics methods at single-cell resolution will significantly enable a deeper understanding of cell type-specific gene regulation, cell diversity and heterogeneity (6). Cost and throughput optimization will also play a crucial role, as making LRS technologies more cost-effective and scalable will accelerate epigenomics research and increase its accessibility to more researchers.

Long-read sequencing is redefining our ability to map and analyze complex epigenetic landscapes, surpassing the limitations of small-read sequencing methods. As computational and experimental techniques continue to evolve, LRS is set to become an indispensable tool for understanding the complex regulatory networks that govern gene expression. This advancement could revolutionize personalized medicine, developmental biology, and understanding disease mechanisms.

References

1.          Liu T, Conesa A. Profiling the epigenome using long-read sequencing. Nature Genetics 2025 57:1 [Internet]. 2025 Jan 8 [cited 2025 Feb 9];57(1):27–41. Available from: https://www.nature.com/articles/s41588-024-02038-5

2.          Altemose N, Maslan A, Smith OK, Sundararajan K, Brown RR, Mishra R, et al. DiMeLo-seq: a long-read, single-molecule method for mapping protein–DNA interactions genome wide. Nature Methods 2022 19:6 [Internet]. 2022 Apr 8 [cited 2025 Feb 9];19(6):711–23. Available from: https://www.nature.com/articles/s41592-022-01475-6

3.          Yue X, Xie Z, Li M, Wang K, Li X, Zhang X, et al. Simultaneous profiling of histone modifications and DNA methylation via nanopore sequencing. Nature Communications 2022 13:1 [Internet]. 2022 Dec 24 [cited 2025 Feb 9];13(1):1–14. Available from: https://www.nature.com/articles/s41467-022-35650-2

4.          Zhong JY, Niu L, Lin Z Bin, Bai X, Chen Y, Luo F, et al. High-throughput Pore-C reveals the single-allele topology and cell type-specificity of 3D genome folding. Nature Communications 2023 14:1 [Internet]. 2023 Mar 6 [cited 2025 Feb 10];14(1):1–18. Available from: https://www.nature.com/articles/s41467-023-36899-x

5.          Zhou T, Zhang R, Jia D, Doty RT, Munday AD, Gao D, et al. GAGE-seq concurrently profiles multiscale 3D genome organization and gene expression in single cells. Nature Genetics 2024 56:8 [Internet]. 2024 May 14 [cited 2025 Feb 9];56(8):1701–11. Available from: https://www.nature.com/articles/s41588-024-01745-3

6.          Lin J, Xue X, Wang Y, Zhou Y, Wu J, Xie H, et al. scNanoCOOL-seq: a long-read single-cell sequencing method for multi-omics profiling within individual cells. Cell Research 2023 33:11 [Internet]. 2023 Sep 12 [cited 2025 Feb 9];33(11):879–82. Available from: https://www.nature.com/articles/s41422-023-00873-5

7.          Kan RL, Chen J, Sallam T. Crosstalk between epitranscriptomic and epigenetic mechanisms in gene regulation. 2021 [cited 2025 Feb 9]; Available from: https://doi.org/10.1016/j.tig.2021.06.014

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