Tom Charlesworth, Director of Marketing Strategy and Corporate Development at biomodal examines how 6-base sequencing can unlock advancements in precision medicine. 

Call it precision medicine, Genomics 3.0, or the age of multi-omics. However you choose to describe it, the underlying meaning is the same: Our ability to collect and analyse molecular data is improving, moving us into a new era where the mechanics of health and disease are rendered with ever more detail. We know that maladies like cancer and Alzheimer’s disease have a genetic component, but advances in molecular biology have highlighted that DNA is just one piece in a much larger pathological puzzle. Identifying the other pieces of this puzzle will improve our understanding of disease, as well as our ability to treat it.  

To this end, 6-base sequencing technology is bringing a key piece of the puzzle into the spotlight. This technology enables researchers to determine a DNA strand’s base sequence while simultaneously annotating the presence of two influential epigenetic marks: 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Known as DNA’s 5th and 6th bases, respectively, 5mC and 5hmC can be found throughout the genome where they exert significant influence over cell behaviour by regulating gene expression. Notably, mounting evidence suggests these bases form distinct, cell-type specific patterns that both reflect and direct cellular identity, sometimes to pathological ends.  

It is increasingly clear that the formation of 5mC and 5hmC patterns represents a significant (epigenetic) piece of the pathological puzzle for many diseases. Accordingly, there is now an effort underway to document the dynamic formation and shifting of these patterns, with many hoping to uncover novel disease mechanisms and expose potential biomarkers. Among those who stand to benefit from these studies are drug developers, who are increasingly in need of sensitive biomarkers. Such work won’t be easy. It will require advanced technology that is capable of reliably parsing 5mC from 5hmC. In other words, researchers need technology capable of 6-base sequencing. 

6-Base Sequencing Exposes The Duality of DNA Methylation 

At a high level, 6-base sequencing technology allows researchers to simultaneously investigate both genetic and epigenetic features of the human genome. In so doing, the technology shines a spotlight on the relationship between DNA and the systems that regulate it, specifically DNA methylation. 

Briefly, DNA methylation is the process of adding a methyl group to cytosine bases (thereby forming 5mC). While this doesn’t change cytosine’s ability to pair with guanine, it does alter how that sequence of DNA interacts with transcription factors and other proteins. Therefore, when 5mC is formed near (or in) a gene, it can either increase or decrease that gene’s expression. By selectively forming 5mC throughout the genome, cells can fine tune gene expression and, by extension, mold their identity. 

However, 5mC is not a permanent base. Just as methyl groups can be added to DNA to elicit an effect, they can be removed to reverse it. The removal of 5mC is a multistep process that can have profound effects on gene expression. Typically it begins with the conversion of 5mC to 5hmC. Though similar, 5hmC has unique physical properties that further alter how cytosine interacts with and recruits DNA binding proteins. Consequently, 5hmC has a distinct influence over gene expression that can greatly differ from that of  5mC. Accordingly, 5mC and 5hmC tend to have opposing effects.  

Given these differences, it is necessary to differentiate between 5mC and 5hmC when studying how DNA methylation contributes to disease development and progression.  

It is only in recent years that we’ve been able to reliably distinguish between 5mC and 5hmC. Much of what we know about DNA methylation was discovered using crude sequencing techniques that fail to differentiate between the two base modifications. Instead, they provide a readout of total modified cytosine levels, combining 5mC and 5hmC signals into one. Though imperfect, these techniques allowed us to identify associations between cytosine modification and gene expression on a global scale (such as hypermethylation being correlated with repressed gene expression). They’ve also helped us recognise that DNA methylation patterns change as cells transition towards disease.  

Yet, the inability to differentiate between 5mC and 5hmC has led to inconsistent, misleading, and contradictory findings, leaving the field with plenty of generalities but few details. If DNA methylation is to become a valuable biomarker, better, more informative technology is needed.  

This is why the advent of 6-base sequencing is so exciting to those studying human disease. By enabling the high resolution detection of 5mC and 5hmC, 6-base sequencing is helping researchers develop a detailed understanding of epigenetic patterns across various disease contexts,1,2,3. This, in turn, is emphasising their potential as valuable biomarkers for myriad applications, including therapeutic development.  

Potential of 6-Base Sequencing in Drug Development 

A growing body of evidence suggests that 5mC and 5hmC patterns—both individually and together—will be valuable biomarkers for therapeutic development, with the potential to refine patient diagnoses, support early and ongoing therapeutic response monitoring, and even guide target discovery. Most of this evidence comes from studies focused on identifying better diagnostic and prognostic biomarkers in cancer.  

Even the most effective therapeutic will fail if given to the wrong patients. Tamoxifen, for example—a popular therapeutic used to treat breast cancer—is only effective in patients whose tumours express oestrogen receptors4. Had this drug been tested in patients without estrogen receptor positive tumours, it would likely have failed. Therefore, when developing highly targeted therapeutics, clinical trials must be designed to ensure the test population can effectively demonstrate therapeutic effect (or lack thereof). This means ensuring patients are accurately diagnosed and subtyped.  

To this end, 5mC and 5hmC show promise. Several analyses using circulating cell free DNA (cfDNA) have shown 5hmC patterns to be a sensitive and specific biomarker, capable of differentiating healthy patients from those with various cancer types, including lung, colorectal, oesophageal, hepatocellular, glioblastoma, and haematological cancers5. Notably, 6-base sequencing was recently used to show that analysis of both 5hmC and 5mC patterns enabled more sensitive detection of tumour-derived cfDNA and therefore early detection of colorectal cancer, outperforming biomarkers that considered total cytosine modification as a single readout, as well those only looking at each methylation mark individually1

Such results suggest that 6-base sequencing can be used to qualify patients for clinical trials by supporting disease diagnosis and subtyping. It should be noted, too, that disease subtyping may be particularly valuable when analyzing discordant clinical trial results, as patients may respond to therapeutics differently depending on their tumor makeup. Here, too, 5hmC has been shown to have biomarker potential: a recent study demonstrated that 5hmC patterns in non-small cell lung cancer were predictive of anti-PD1 therapeutic response6.

The ability to sensitively detect disease using 5mC and 5hmC levels in cfDNA may also help to accelerate clinical trial completion. For instance, a reduction in tumour-derived cfDNA (identified by its distinct 5mC and 5hmC patterns) can be an early indicator of therapeutic response. 

Similarly, changes in 5mC and 5hmC patterns may indicate growing therapeutic resistance6,7 Monitoring patient response using epigenetic biomarkers may help researchers identify successes and failures faster.  

Lastly, studying the relationship between 5hmC and 5mC in disease has the potential to reveal novel therapeutic strategies. In its simplest use case, identifying how abnormal methylation patterns contribute to disease may reveal subtle drivers of oncogenic phenotypes which hold therapeutic value. Additionally, tools such as CRISPR on/off can be used to modify DNA methylation at specific genes8. One can imagine developing a therapeutic that compensates for abnormal, oncogenic methylation patterns with CRISPR on/off.  

New Eras Demand New Technology 

Decades of study have made it clear that the epigenome is critically involved in disease pathogenesis. DNA methylation has been shown to influence disease development, progression, and therapeutic response. Yet, our understanding of DNA methylation has been limited to generalities riddled with exceptions. This is thanks, in large part, to the limitations of standard methylation sequencing technologies that fail to distinguish between 5mC and 5hmC, two common and functionally dichotomous cytosine modifications.  

The advent of 6-base sequencing will not solve all of the mysteries surrounding the epigenome. But it does hold significant potential to demystify DNA methylation and its relationship to disease mechanics. In doing so, 6-base sequencing may help validate 5mC and 5hmC as valuable biomarkers with applications not only in basic research, but in diagnostics, prognostics, and drug development. Gone are the days of vague methylation studies. This is the era of 6-base sequencing.  

About the author  

Tom Charlesworth, PhD is the Director of Marketing Strategy and Corporate Development at biomodal. Tom has a wealth of experience defining strategy for biomodal’s products. His focus on customer needs, market trends, and key partnerships supported the launch of duet multiomics solutions, revealing the power of the 5- and 6-base genome.  

References 

  1. Puddu, F. et al. (2024) 5-methylcytosine and 5-hydroxymethylcytosine are synergistic biomarkers for early detection of colorectal cancer [Preprint]. doi:10.1101/2024.10.30.621123 
  2. Guerin, LN et al. (2025) Temporally discordant chromatin accessibility and DNA demethylation define short- and long-term enhancer regulation during cell fate specification. Cell Rep. https://doi.org/10.1016/j.celrep.2025.115680 
  3. Hardwick, JS et al. (2025) SCoTCH-seq reveals that 5-hydroxymethylcytosine encodes regulatory information across DNA strands. PNAS. https://doi.org/10.1073/pnas.251220412 
  4. American Cancer Society (2021) Breast cancer hormone receptor status [online]. Available at: https://www.cancer.org/cancer/types/breast-cancer/understanding-a-breast-cancer-diagnosis/breast-cancer-hormone-receptor-status.html (Accessed: 15 October 2025). 
  5. Song, D. et al. (2025) 5-Hydroxymethylcytosine modifications in circulating cell-free DNA: frontiers of cancer detection, monitoring, and prognostic evaluation. Biomarker Res. 13:39. https://doi.org/10.1186/s40364-025-00751-9  
  6. Guler, G. D. et al. (2024) Plasma cell-free DNA hydroxymethylation profiling reveals anti-PD-1 treatment response and resistance biology in non-small cell lung cancer. J. Immunother. Cancer 12: e008028. https://doi.org/10.1136/jitc-2023-008028  
  7. Menghi, F. et al. (2022) Genomic and epigenomic BRCA alterations predict adaptive resistance and response to platinum-based therapy in patients with triple-negative breast and ovarian carcinomas. Sci. Transl. Med. 14:eabn1926. https://doi.org/10.1126/scitranslmed.abn1926  
  8. Sproul, D. et al. (2016) Cell-type-specific mapping of the human epigenome reveals factors shaping DNA methylation across tissues. Genome Biol. 17: 151. https://doi.org/10.1186/s13059-016-1011-x