Every cell in the human body contains nearly identical DNA, yet cellular behavior varies significantly. The danger posed by cancer cells arises not only from the presence of mutations but also from the resulting changes in gene expression, protein production, and ultimately, cellular behavior. Understanding the connection between genetic mutation and cellular behavior remains one of biology’s most challenging problems.
A central challenge is to determine how specific DNA variants alter cellular function, which requires simultaneous analysis of both DNA and RNA, the molecular indicator of gene activity, in the same cell. Until recently, achieving this accurately and at scale was essentially impossible.
A new method, SDR-seq (single-cell DNA–RNA sequencing), developed by researchers at the European Molecular Biology Laboratory (EMBL) and Stanford University, addresses this challenge for the first time at high throughput. Published in Nature Methods in October 2025, this technique represents a significant breakthrough in studying the relationship between genetic variation and disease.
Why Reading DNA and RNA Together Is So Hard
To appreciate the significance of SDR-seq, it is important to consider the limitations of previous approaches. Single-cell RNA sequencing has been available for years, providing information about gene activity. Similarly, they have also identified single-cell mutations. However, obtaining accurate, high-quality measurements of both DNA and RNA from the same cell simultaneously has proved challenging. The primary technical obstacle is allelic dropout (ADO), which occurs when one or both copies of a gene at a specific genomic position are not detected. Existing high-throughput methods for simultaneous DNA and RNA measurement exhibited ADO rates exceeding 96%. Consequently, at any given genomic position, these tests failed to detect either DNA copy in more than 96% of cases. This limitation made it nearly impossible to accurately determine whether a variant was present in one or both copies, a distinction critical for understanding its impact on cellular function.Understanding how it affects the cell.
SDR-seq achieves accurate allele detection in approximately 90% of cells, substantially improving reliability and making the method practical for addressing complex biological and medical questions.
How SDR-seq Actually Works
The method is conceptually straightforward yet technically sophisticated in its execution.
Cells are initially fixed and chemically preserved, then subjected to in situ reverse transcription, which converts active RNA transcripts into stable DNA copies (cDNA) within the intact cell. The key innovation is performing this RNA copying step prior to any other manipulation, thereby preserving both DNA and RNA information within each cell.
Cells are then loaded into a droplet-based microfluidic system (using Mission Bio’s Tapestri technology). Each individual cell receives its own tiny droplet, which is lysed and subjected to a multiplexed PCR reaction that amplifies up to 480 specific DNA locations and RNA targets of interest. Each droplet also receives a unique barcode, so every resulting sequence can be tracked. The resulting DNA and RNA libraries are sequenced separately, enabling optimized coverage for each. This process yields a comprehensive map across thousands of cells simultaneously, indicating which genetic variants are present and which genes are active in each cell. A key practical advantage of SDR-seq is its targeted approach, in contrast to competing methods that require whole-genome sequencing of each cell, which is both costly and yields sparse data. Researchers can design panels targeting specific genomic loci and genes of interest, achieving deep and reliable coverage of these targets without expending sequencing resources on irrelevant regions.
How SDR-seq Actually Works
The method is simple in theory, but it requires advanced techniques to implement.
First, cells are fixed and preserved with chemicals. Then, in situ reverse transcription turns the cell’s active RNA into stable DNA copies (cDNA) inside the cell. The main innovation is doing this RNA copying step first, which keeps both DNA and RNA information safe in each cell.
Next, the cells are placed into a droplet-based microfluidic system (using Tapestri technology from Mission Bio). Each cell is placed into its own droplet, where it is lysed and undergoes a multiplexed PCR reaction that can amplify up to 480 specific DNA and RNA targets at once. Each droplet gets a unique barcode, so every sequence can be traced back to its original cell.
The DNA and RNA libraries generated by this process are sequenced separately, which provides better coverage for each. This results in a detailed map for thousands of cells at once, showing which genetic variants are present and which genes are active in each cell.
A key practical advantage is that, unlike other methods that require sequencing the whole genome of each cell, which is expensive and gives less data, SDR-seq uses a targeted approach. Researchers can focus on specific genes and regions, obtaining deep, reliable results without wasting resources on unimportant areas.
What the Team Demonstrated: Three Powerful Applications
The researchers did more than just create the tool; they showed it can solve real problems in biology and disease.
Testing in stem cells: The team first tested SDR-seq in human induced pluripotent stem cells (iPSCs), which are lab-grown cells that can mimic many types of human cells. They showed that SDR-seq could handle panels of 120 to 480 DNA and RNA targets simultaneously while remaining highly reliable. Importantly, the method worked well regardless of where the target was in the genome,, whether in active regions, regulatory elements, or other areas.
Detecting gene expression changes from variants: A key question in genomics is whether a specific DNA variant alters a gene’s function. The team used SDR-seq with CRISPR-based editing tools to test dozens of variants called expression quantitative trait loci (eQTLs), which are linked to changes in gene activity in people. They could spot even small changes in gene expression caused by certain variant combinations, including in the 3′ untranslated region of POU5F1, a gene important for stem cell identity. The method was sensitive enough to distinguish the effects of different nearby variants, with a level of detail that older methods could not achieve.
Revealing cancer biology in real patient tumors: The most striking use was in B-cell lymphoma, a cancer of the immune system’s B cells. The team studied tumor samples from three patients with different types of B-cell lymphoma, analyzing 3,600 to 8,400 cells per sample for genetic mutations and gene expression simultaneously.
The results were eye-opening. Inside tumors, cells are at different stages of development. Some are in an early stage, the dark zone (DZ), while others are in a later stage, the light zone (LZ). Using SDR-seq, the researchers found that cells with more mutations were predominantly in the light zone. These cells also showed higher activity in B-cell receptor signaling and in genes that help tumors survive and evade cell death. In short, the more mutations a cancer cell had, the more it activated survival signals. SDR-seq allowed scientists to see this link directly, cell by cell, for the first time.
Why This Changes the Game for Medicine
SDR-seq’s impact goes far beyond just one experiment.
The vast majority, over 90% of genetic variants associated with common human diseases through genome-wide association studies, are located in the noncoding genome: the regions of DNA that don’t directly encode proteins but regulate when and how much of each gene gets made. These regulatory variants have been extraordinarily difficult to study because existing tools couldn’t reliably link them to changes in gene expression. In contrast, natural cSDR-seq can detect noncoding variants and link them to changes in gene expression within the same cell. It can also do this at scale, with thousands of cells and hundreds of targets in a single experiment.
This opens the door to systematically understanding the regulatory logic of the genome, identifying which noncoding variants matter, what they do, and why they contribute to diseases such as cancer, autoimmune conditions, heart disease, and neurological disorders.
For cancer, connecting mutation burden to cell behavior in real patient tumors, not just lab models, helps scientists better understand how tumors evolve, why some tumors resist drugs, and which cancer cells are most dangerous. Tumor cells with more mutations and higher survival signals are often the ones that escape treatment. SDR-seq enables visualization and identification of these cells, and perhaps even targeting them.
What Comes Next
The researchers are clear about both the power and the current limits of SDR. The researchers are open about both the strengths and current limits of SDR-seq. The method examines specific regions rather than the whole genome, so it must be configured to focus on known variants rather than to discover entirely new mutations. Also, the primary editing tools used in some experiments were not very efficient, making it harder to interpret some results. Targeting the mitochondrial genome could enable tracing cell lineages, tracking how individual cells and their descendants evolve over time. And enhanced RNA readout strategies might eventually enable whole-transcriptome profiling alongside the targeted DNA panel.
The code and data pipeline (SDRranger) are publicly available, and a detailed protocol has been published, making the method accessible to research groups worldwide.
The Bottom Line
SDR-seq achieves something that has been out of reach for years: it simultaneously, accurately, and at scale determines both the genetic identity and functional state of thousands of individual cells. It bridges the gap between the genome, the static instruction manual, and the transcriptome, the dynamic record of which instructions each cell is actually using.
To understand how mutations drive cancer, how noncoding variants affect disease, and how individual cells differ from their neighbors in the same tissue, scientists have been waiting for this tool. Published in Nature Methods by researchers from EMBL Heidelberg and Stanford University, SDR-seq is a real step forward in understanding what happens inside living cells, one cell at a time.