The pioneer of single-cell sequencing
Dr. Ashley Sanders takes a seat at the head of the table, her usual spot during her weekly lab meetings. Marcella Franco, a doctoral student, has just processed sequencing data on her first patient sample – cells from a patient with the autoimmune disease Lupus – and is presenting it to Sanders’ lab group.
To the casual observer, the data looks like abstract art – patterns of colored lines and clusters of vibrant dots. But they represent a potential goldmine of information. It is the first time that anyone has looked for structural variants – deletions, inversions, duplications or translocations – in the DNA of individual cells from a person with Lupus. Franco hopes to identify variants that might be contributing to the disease.
While the concept of how genetic mutations develop and promote cancer is well known, the idea has been poorly studied in other types of diseases. Such insights might help us develop single-cell approaches to personalized medicine.
“The unknown is the space we are working in,” says Sanders, Group Leader at the Berlin Institute for Medical Systems Biology of the Max Delbrück Center (MDC-BIMSB).
Sanders is a pioneer in the field of single-cell sequencing. She helped develop new technology to study the human genome, which is overturning long held dogmas about what a “normal” genome looks like. It is also raising intriguing questions about whether mutations in somatic cells – cells that develop after conception and are therefore not inherited or passed on – contribute to diseases other than cancer, particularly inflammatory diseases.
Her research suggests that a diseased environment, such as inflammation, may favor cells with specific types of genomic variations, called subclones. And as these subclones proliferate, they may contribute to disease progression in a kind of feedback loop. They may also modify how cells respond to environmental stimuli or even treatment. "While the concept of how genetic mutations develop and promote cancer is well known, the idea has been poorly studied in other types of diseases,” Sanders says. “Such insights might help us develop single-cell approaches to personalized medicine.”
New tools to study individual cells
Over the past decade, single-cell sequencing has come of age. Between 2012 and 2017, the U.S. National Institutes of Health established a Common Fund program to support research in single-cell genomics. The funding enabled researchers to develop new and unique tools to study individual cells in ways that were previously not possible.
Most researchers have been using the technology to analyze the RNA in individual cells, likely because RNA is more abundant than DNA and easier to capture, says Sanders. There can be thousands of copies of an RNA transcript in a single cell, plus they all share a common RNA sequence, which makes them easier to extract.
By contrast, DNA exists as only two copies in a single cell (one maternal and one paternal copy) and is much harder to process than RNA. Consequently, hardly anyone has been using single-cell sequencing to study DNA, says Sanders. Moreover, since it was assumed all of a person’s cells carry the exact same DNA sequence, not many people understood the point of it, she adds. Thus, single-cell sequencing of DNA remained a backwater endeavor until relatively recently.
A new technique is born
Sanders’ focus on single-cell DNA rearrangements came about almost by accident. While pursuing her doctoral thesis at the University of British Columbia with Professor Peter Lansdorp, she noticed that a single-cell DNA technique she was using could identify genomic inversions – large segments of the genome where the DNA sequence has flipped in orientation. But she never thought it would lead to anything – she didn’t think anyone cared about inversions, she says.
Microfluidic Microplate Dispenser – scientific device for single cell sequencing
In 2016, Professor Jan Korbel, Senior Scientist and Head of Data Science at the European Molecular Biology Laboratory in Heidelberg, heard Sanders present her data at a meeting. He instantly saw the potential.
“I was intrigued,” says Korbel. “I thought it could do more than just detect inversions. It was very cool.”
At the time, Korbel was chair of the Human Structural Variation Consortium. The consortium had invested a lot of effort on finding inversions, but with little success, Sanders says. “When I showed up with my little technology and was like ‘by the way, we can find inversions by accident’ they got really excited. That was really a turning point for my career.”
Korbel later asked Sanders to join his lab as a post-doc. Together with a team at the EMBL, they honed Sander’s single-cell DNA sequencing technique and developed Strand-seq to discover and characterize all types of structural variants in single cells. “Sometimes I joke that, there were five people in the world who cared about inversions and I was able to find them,” Sanders quips.
How it works
The majority of available genetic data is an average taken from sequencing millions of cells, Sanders explains. But this type of DNA sequencing, called bulk sequencing, isn’t sensitive enough to pick up most types of DNA rearrangements, especially inversions.
Strand-seq takes a different approach. Unlike bulk sequencing that provides sequence data from a mixture of cells and DNA strands, Strand-seq takes advantage of the directionality of DNA and sequences each strand of DNA’s double helix for each cell. Having information from both copies of DNA from every single cell gives researchers more certainty that any variation detected is correct.
Imagine a row of differently colored Lego blocks in which the order of colors matches exactly what is given in the instruction manual – except for one block. Did the person who built the row make a mistake? Or did they place that block in that position on purpose? One could not answer the question without asking the person. Now suppose the same person built a complementary row of Lego blocks in the exact opposite order. If the same block does not match what is shown in the manual, one can be more certain that both blocks were placed there on purpose.
Since other technologies don’t sequence both strands of complementary DNA, researchers can’t say with certainty whether a variation they find is real or an error, explains Korbel. With Strand-seq we have double verification, he says. “We see signatures that are unlikely to be random events.”
New insight into genomic variation
The technology has already turned up surprises. Sanders and Korbel recently co-authored a paper published in “Nature Genetics,” for example, that found somatic structural variants in one in every 40 blood cells of healthy people. It has always been thought that every cell in the body carries the same DNA – any deviation was considered abnormal and a hallmark of disease. But that structural variants were found in the cells of apparently healthy people suggests that genomic mosaicism is far more common than anyone previously thought; it may just be part of normal genetic variation.
However, no-one knows for sure what the finding means yet. Although people over the age of 60 tended to have greater numbers of cells with structural variants, one study participant who was over 90 years old had no structural variants in her sample, says Sanders. What’s more, the study found that the chance of a mutation arising in blood cells was the same, regardless of age.
“That was really shocking to me,” says Sanders. “The assumption has always been that the risk of mutations increases as we age.”
Unexpected success
Sanders never expected to complete any type of higher education, let alone become a scientist. The native Canadian is the first person in her family to have completed high-school. “The expectations were never there, not even from myself,” Sanders says. “I have always just stayed open and curious. I am not afraid of taking on a new challenge, even when I feel way out of my depth.”
My goal is to create knowledge that benefits society. The best way to do that is by working together and as a team. Knowledge is what drives us, and unites us. And we all have so much fun in the lab.
She is stunned at how far the field has progressed over the past decade. Ten years ago, Sanders got puzzled looks from her peers because they didn’t understand the point of sequencing single cells. Today, “Strand-seq has transformed the structural variation detection field overall,” she says. The technique is being applied in other research fields, species conservation and evolutionary biology for example. It can also be used to improve the accuracy of other genomic methods, and now, Sanders hopes, to help researchers better understand how inflammatory diseases, such as Lupus, develop and progress.
During her lab meeting, Sanders offers Franco tips on how to present her data. With only a few samples from Lupus patients thus far analyzed, it’s too early to draw any conclusions about whether single-cell DNA rearrangements contribute to the disease.
But Sanders seems just as focused on the students as on the data. She chose to work in research instead of industry because she was drawn to the idea of mentoring. “I want to create great scientists, and contribute to future generations” she says. She herself had a beloved mentor, Ester Falconer, who always told her to “just pay it forward,” whenever Sanders expressed appreciation. Of course, she also wants to do great science, she adds, but her end game is not acclaim or fame:
“My goal is to create knowledge that benefits society. The best way to do that is by working together and as a team. Knowledge is what drives us, and unites us. And we all have so much fun in the lab.”
Text: Gunjan Sinha
