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As next-generation sequencing swept across much of biology, scientists collected enormous amounts of information on the strings of bases—adenine, guanine, cytosine, and thymine—that make up DNA, but key information was often neglected. Although next-gen platforms simplified sequencing an organism’s genetics, technical hurdles slowed down the collection of epigenetic information. As Tom Charlesworth, PhD, director of market strategy and corporate development at biomodal, explains, “Epigenetics is sort of the regulatory layer of the genome that decides what genes are switched on and off in the different cell types of tissues.” For a comprehensive view of basic biology and the mechanisms behind disease, scientists need an easier way to combine genetic and epigenetic information.
Traditional sequencing gathers the order of the DNA bases, but it misses epigenetic features in DNA. “Methylation is an important mechanism by which epigenetic regulation is implemented,” says Charlesworth. “The methylation message happens at cytosines.” Those bases can be unmethylated or methylated in two forms: 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), which are generally understood to turn genes off and on, respectively. These methylation marks change in response to a person’s environment. That is, the state of methylation acts as the layer in which the environment can affect what a person’s cells and tissues are actually doing inside the body.
If scientists could easily combine genetic and epigenetic information, the results could be used to transform the clinical approach to diseases, from diagnosis and tracking disease progression and recurrence to developing new treatments or even predicting and preventing diseases. Scientists at biomodal developed a solution that will support researchers in their mission to make all of this possible, as well as delving deeper into the mechanisms of many biological processes.
From four letters to five
Conventional methods used to capture genetic and epigenetic information from a sample require multiple processes: genetic sequencing; detecting methylation of cytosines; and complicated bioinformatic steps to combine the data. The overall complexity of these workflows limits their uptake and utility.
Beyond the complexity, the process could even confound the data. Bisulfite treatment to detect methylation is known to damage DNA, plus some of the chemistry used to distinguish unmethylated from methylated cytosines can cause these bases to be erroneously read as thymines. Consequently, cytosine-to-thymine mutations—one of the most common mutations in cancer—can be missed. Plus, method-induced cytosine-to-thymine changes reduce the accuracy of mapping a genome.
biomodal—known as Cambridge Epigenetix until April 2023—simplified these steps with five-letter sequencing, which determines a sample’s sequence and detects cytosine methylation. biomodal’s five-letter sequencing product, duet multiomics solution +modC, uses a hairpin technology that captures the original sequence and a copy in one molecule, and it includes the software required to integrate the information and perform error correction. Then, the process correctly reads the bases—A, G, C, or T—and detects methylated cytosines.
Simplified steps to six-letter sequencing
However, with methylated cytosine readouts typically conflating 5mC and 5hmC, and 5mC generally repressing a gene’s expression while 5hmC promotes it, the ability to distinguish the two will be a powerful tool supporting researchers’ efforts to understand, detect, and treat disease. “Imagine what you’re reading is like a stoplight where, instead of seeing red or green, you’re always seeing some sort of yellow,” says Kurt Yardley, PhD, director of product marketing at biomodal. “You can’t tell if a gene should be going or stopping.”
Recently, a group of scientists from biomodal described six-letter sequencing.1 As these scientists explained: “We present a single base-resolution sequencing methodology that sequences complete genetics and the two most common cytosine modifications in a single workflow.” They added that the method had been “demonstrated on human genomic DNA and cell-free DNA from a blood sample of a patient with cancer.” In addition, this method works with as little as five nanograms of sample. As these experts from biomodal concluded: “Simultaneous, resolved reading of genetic and epigenetic bases provides a more complete picture of the information stored in genomes and has applications throughout biomedicine.”
biomodal’s new product, duet multiomics solution evoC, measures multiple dimensions of a single sample in one experiment, enabling scientists to elucidate disease mechanisms, biological phenotypes, and discover new insights. In short, this product provides standard four-letter sequencing—A, G, C, and T—and distinguishes between 5mC and 5hmC, accurately detects cytosine-to-thymine mutations and can even be used to predict gene expression, chromatin accessibility, and enhancer state. Although other technologies that try to do multiple things at once always lose information, duet evoC maintains accuracy across all of the data types collected.
Scientists also get more information from duet evo. Instead of being stuck at Yardley’s ambiguous stoplight, scientists get a clear picture of the traffic control going on with the combination of genetics and epigenetics. “It adds a level of dimensionality,” Yardley says. “What you’re seeing with duet evoC is not a static picture.” Instead, duet evoC portrays a system in action. Beyond picking out 5mC’s and 5hmC’s, duet evoC also detects the progression between these states of methylation at key gene regulatory sites (enhancers)—essentially predicting the gene-expression transition from off to on. “So, it’s time traveling,” Charlesworth says. “It’s predicting the future.”
Today’s scientists use multiple methods to study and understand gene expression, including RNA sequencing (RNA-seq) and chromatin accessibility. RNA-seq, however, often combines RNA that has been produced plus the RNA that is being produced, the so-called nascent RNA. By comparison, information about the methylation of cytosines can tell us which genes are currently being expressed and even ones primed for expression, whilst simultaneously providing information about chromatin accessibility. With the added information delivered by duet evoC, scientists can more accurately study functional genomics, which connects a person’s genotype and phenotype.
In addition to duet evoC’s powerful production of data, it’s easy to use. The method is very similar to what people are already using, so it fits easily into current workflows. The bioinformatic software significantly simplifies data analysis and the extraction of useful information from that data.
To the brain and beyond
Scientists at biomodal already envision applying duet evoC in cancer research and the development of new therapies, because 5mC and 5hmC profiles are often altered differently in cancer. In preliminary studies using cell-free DNA from patients with colorectal cancer, for example, Charlesworth points out that “by separating the 5mC and 5hmC rather than reading the conflated signal, you can see examples where enhancers go from repressed, to primed, to active, and can measure the methylation differences between healthy individuals and stage I patients at these enhancer regions. In addition, duet evoC could add a critical dimension when monitoring treatment response and potential recurrence by testing for minimal residual disease.”
The brain could also be a crucial area of applying duet evoC. Scientists already know that 5hmC levels are 10-fold higher in the brain than anywhere else. Various stimuli and the progression of neurodegenerative diseases also correlate with distinct changes in both 5mC and 5hmC patterns in brain cells. Elucidating these signals with duet evoC from a single low-input sample could be the technological leap needed for the next scientific breakthrough.
Really, the range of applying duet evoC appears unlimited. Only when scientists around the world start putting this solution to work will its full potential start to emerge. When the biomodal team introduce scientists to data from duet evoC, they envision ways to apply it. “You can see it in their eyes,” Charlesworth says. “They’re thinking about the possibilities of how they can use it.”
Unlimited and exciting, the full potential of duet evoC comes as no surprise. “It lets you disentangle the biology of what’s going on,” Charlesworth says. “Then, you can better understand the role of 5mC and 5hmC in health and disease, what’s being activated or repressed within the genome, and what’s happening in functional genomics.”
- Füllgrabe, J., Gosal, W.S., Creed. P., et al. Simultaneous sequencing of genetic and epigenetic bases in DNA. Nature Biotechnology 41:1457–1464. (2023).
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