Epigenetics is all about understanding how gene activity can change without altering the DNA sequence itself. Think of it as a “volume control” for your genes, turning them up or down based on environmental and cellular signals. These adjustments are driven by mechanisms like DNA methylation, chromatin architecture, and non-coding RNA activity. Epigenetics plays vital roles in how cells function and can impact everything from development to disease, especially in conditions like cancer1. Let’s dive into how epigenetics works and how HiFi sequencing technology is changing the game.
Understanding DNA methylation
DNA methylation refers to the addition of a methyl group to one of the four nucleotide bases in DNA2 —essentially marking it for specific functions. For one of the most common types of methylation, the methyl group attaches to the 5th carbon molecule of a cytosine base, leading to the designation 5mC methylation3. Methylation is crucial for human development and is influenced by environmental factors like diet or stress. When things go awry, such as abnormal methylation patterns, it can lead to diseases like cancer, where tumor suppressor genes might get switched off4.
Bisulfite conversion, which converts methylation status into a base change, has been used to study methylation with sequencing or microarrays, but this approach can degrade DNA, making it tough to get a full picture. PacBio HiFi sequencing offers a more accurate, comprehensive view of methylation automatically built into every run without the need for bisulfite treatment or additional library preparation methods5, enabling researchers to easily integrate methylation insights into a broader understanding of epigenetic regulation.
Chromatin architecture: The structural basis of epigenetics
Chromatin architecture encompasses the structural organization of how DNA can be tightly packed into the nucleus of a cell. Chromatin fibers are long strands of DNA wrapped around proteins called histones. Together these structures resemble beads on a string called nucleosomes, and play a pivotal role in packaging DNA and regulating its accessibility. This dynamic structure influences how genes are expressed by controlling whether specific DNA segments are open for transcription or tightly packed and inaccessible.
A groundbreaking method called Fiber-seq uses HiFi sequencing to identify chromatin architecture including the positioning of nucleosomes, transcription factors, and regions of accessible chromatin along each read6. By examining chromatin architecture, researchers can understand how the spatial arrangement of DNA and associated proteins impact gene regulation.
A new era in epigenetic research
In a previous blog, we described the power of combining Fiber-seq with methylome sequencing in addition to the genome and transcriptome on a single Revio SMRT Cell from a preprint led by Dr. Andrew Stergachis and Dr. Mitchell Vollger at the University of Washington. Today’s blog now celebrates the official publication of this method just yesterday in Nature Genetics. The importance of this landmark publication bears repeating – by combining four high-quality, haplotype-resolved ‘omes – the genome, CpG methylome, chromatin epigenome, and transcriptome into a single run, this method fast tracks the ability to resolve the mechanisms underlying unexplained rare diseases. This achievement demonstrates the potential of PacBio technology to provide a holistic view of gene regulation, offering exceptional insights into complex biological processes. The study highlights how combining these layers of information can deepen our understanding of diseases and lead to more effective diagnostics and treatments.
Exploring the complex epigenetic landscape
Beyond DNA methylation and chromatin architecture, a variety of other mechanisms contribute to the intricate regulation of gene expression. Non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), are crucial players in fine-tuning gene activity. For example, miRNAs typically function by binding to messenger RNAs (mRNAs), preventing their translation into proteins. LncRNAs, on the other hand, can influence gene expression by interacting with chromatin or by guiding regulatory proteins to specific genomic regions. These ncRNAs have emerged as key regulators in processes like development, differentiation, and disease.
Moreover, the process of RNA modification—where the RNA sequence is altered after transcription—adds another layer of complexity to gene regulation. This mechanism can influence how genes are expressed without changing the underlying DNA sequence, offering a dynamic means of controlling gene function in response to environmental or cellular cues.
These diverse mechanisms add complexity to the epigenetic landscape, and highly accurate HiFi long-read sequencing helps map these interactions more clearly by capturing long sequences in their entirety.
HiFi sequencing: A more comprehensive solution for the epigenome
PacBio HiFi sequencing offers a more comprehensive solution for epigenetic profiling by offering a highly accurate, single-molecule view of DNA, RNA, and its associated methylation signatures on one platform. By integrating exceptional 99.9% accuracy and reads of 20 kb or more, this view extends to complex regions of the genome that other technologies are not known to reach, like repetitive regions or structural variants. Unlike other methods, HiFi sequencing captures native molecules without bisulfite treatment or amplification, preserving the original epigenetic marks. This makes it aptly suited to accurately and comprehensively characterize the epigenome. This streamlined approach not only saves time but also ensures the integrity of the DNA, enabling researchers to capture multiple methylation signatures in a single run.
The release of SPRQ chemistry on the Revio system, alongside updates to SMRT Link and instrument software, significantly boosts the multiomics capabilities of Revio runs. This includes improved DNA methylation calling, extending the reach to 6mA calling, a marker of open chromatin in Fiber-seq, and providing greater accuracy for 5mC detection This advancement, alongside a 33% increase in data output enables researchers to capture multiple dimensions of the epigenome on PacBio platforms and revolutionize epigenomic research at scale.
Transformative Impact Across Fields
The ability of HiFi sequencing technology to capture new advances in epigenetics has been transformative across multiple fields of study:
- Cancer Research: HiFi sequencing has pinpointed methylation patterns in the COLO829 melanoma cell line via hypermethylation upstream of KLLN, a p53-regulated DNA replication inhibitor7.
- Rare disease: Atypical methylation patterns contribute to rare diseases like myotonic dystrophy and are important factors in pathogenic repeat expansions, such as the CGG expansion at the FMR1 locus that causes Fragile X syndrome. With high accuracy, long reads, and methylation detection, HiFi sequencing is ideal for characterizing these repeat expansions3.
HiFi sequencing phases and identifies hypermethylation of the region adjacent to a mosaic 5 kb DMPK expansion in a sample with myotonic dystrophy (Children’s Mercy Kansas City). Magenta indicates methylation, while blue indicates unmethylated bases.
- Neuroscience: The role of methylation was examined in 92 Alzheimer’s patients and 117 Healthy centenarians from the Amsterdam Dementia Cohort. Using HiFi sequencing they were able to identify an AluYb8 retrotransposon in the 3’ UTR of the risk Haplotype TMEM106B. Simultaneous methylation information shows TMEM106B haplotypes carrying the ALUYb8 element are more methylated than those without8
- Microbial genomes: The simultaneous detection of methylated DNA bases has resulted in a transformation of microbial research9 and has been extensively applied for the characterization of their effects on pathogenicity10, immune system invasion11, and beyond.
The future of epigenetics
PacBio sequencing is paving the way for a deeper understanding of epigenetic regulation. From cancer breakthroughs to insights into rare diseases, HiFi sequencing is unlocking new possibilities, making it an indispensable tool in modern biology.
Epigenetics holds the key to many biological mysteries, and with tools like HiFi sequencing, we’re just beginning to unravel its full potential. By integrating genetic, epigenetic, and transcriptomic data in a single experiment, the possibilities for discovery are endless. Researchers can now approach long-standing questions with a more holistic perspective, bridging the gap between molecular details and systems-level understanding.
PacBio continues to set new standards for genomic research, driving innovation that shapes the future of epigenetics and beyond. With these advancements, the scientific community is poised to unlock answers to some of the most pressing challenges in global health.
References
- Sharma, S., Kelly, T. K., & Jones, P. A. (2010). Epigenetics in cancer. Carcinogenesis, 31(1), 27-36. https://doi.org/10.1093/carcin/bgp220
- Epigenetics, Health, and Disease. CDC. https://www.cdc.gov/genomics-and-health/epigenetics/index.html Accessed 28 January 2025.
- PacBio Application brief – Measuring DNA methylation with 5-base HiFi sequencing https://www.pacb.com/wp-content/uploads/application-brief-measuring-dna-methylation-with-5-base-hifi-sequencing.pdf
- Alshammari, E., Zhang, Y., Sobota, J., & Yang, Z. (2022). Aberrant DNA methylation of tumor suppressor genes and oncogenes as cancer biomarkers. Genomic and Epigenomic Biomarkers of Toxicology and Disease: Clinical and Therapeutic Actions, 251-271. https://doi.org/10.1002/9781119807704.ch12
- PacBio – Epigenetics https://www.pacb.com/products-and-services/applications/epigenetics/
- Stergachis, A. B., Debo, B. M., Haugen, E., Churchman, L. S., & Stamatoyannopoulos, J. A. (2020). Single-molecule regulatory architectures captured by chromatin fiber sequencing. Science, 368(6498), 1449-1454. https://doi.org/10.1126/science.aaz1646
- PacBio Application note – Robust detection of somatic variatns from tumor-normal samples with highly accurate long-read whole genome sequencing. https://www.pacb.com/wp-content/uploads/Application-note-Robust-detection-of-somatic-variants-from-tumor-normal-samples-with-highly-accurate-long-read-whole-genome-sequencing.pdf
- Salazar, A., et al. (2023). An AluYb8 retrotransposon characterises a risk haplotype of TMEM106B associated in neurodegeneration. medRxiv, 2023-07. https://doi.org/10.1101/2023.07.16.23292721
- Davis, B. M., Chao, M. C., & Waldor, M. K. (2013). Entering the era of bacterial epigenomics with single molecule real time DNA sequencing. Current opinion in microbiology, 16(2), 192-198. https://doi.org/10.1016/j.mib.2013.01.011
- Ailloud, F., Gottschall, W., & Suerbaum, S. (2023). Methylome evolution suggests lineage-dependent selection in the gastric pathogen Helicobacter pylori. Communications Biology, 6(1), 839. https://doi.org/10.1038/s42003-023-05218-x
- Phillips, Z. N., Husna, A. U., Jennings, M. P., Seib, K. L., & Atack, J. M. (2019). Phasevarions of bacterial pathogens–phase-variable epigenetic regulators evolving from restriction–modification systems. Microbiology, 165(9), 917-928. https://doi.org/10.1099/mic.0.000805