By combining two innovative approaches, researchers can now sequence the full spectrum of mutational differences between individual cells’ genomes.
Traditional sequencing is often likened to making a smoothie: researchers blend a bunch of cells, obtain an average sequence, and draw conclusions on the ingredients that comprise the slush. More recently, scientists have gained the ability to perform single-cell sequencing, which can reveal rare variations between cells and the evolution of cell lineages. But current methods require reading the genome in short sections and therefore often fail to capture complex repetitive regions, which scientists are increasingly linking to health and disease. Long-read technologies could overcome this pitfall; however, their methods require much more DNA than can be extracted from a single cell. Single-cell, long-read sequencing has remained frustratingly out of reach.
That is, until now. By combining “two very innovative approaches” a cutting-edge DNA amplification technique with the latest advances in DNA sequencing a team of scientists have applied long-read technology to single cells, says Alexander Hoischen, a researcher of genomic technologies at Radboud University Medical Center in The Netherlands who was not involved in the research. “This was unthinkable just two or three years ago,” says Hoischen.
The feat may allow for a more detailed look at mutations underlying all sorts of diseases, experts tell The Scientist.
Over the past decade, reads the product of DNA sequencing have been getting longer. Long-read sequencing has allowed scientists to sequence troublesome “dark regions” of the genome that are inaccessible to short-read technologies, either due to an abundance of guanines and cytosines, or duplicated regions not easily mapped to a chromosome.
However, long-read sequencing requires a ton of DNA. Several micrograms of genetic material are needed, but “a single cell contains just six picograms,” says Joanna Hård, a computational biologist at ETH Zurich in Switzerland. “So substantial amplification is required before you can sequence it” using long-read methods, she says.
And that’s where things get tricky, says Hård, as the primary methods used to amplify DNA are prone to “amplification bias”: the tendency for certain sequences to be ramped up at the expense of others. Now, Hård and colleagues have obtained long reads from individual cells using an improved DNA amplification method. Though not yet peer-reviewed, the results were reported in a preprint uploaded to bioRxiv on January 23. To minimize amplification bias, the team used a technique called droplet-based multiple displacement amplification. It works by trapping DNA fragments in droplets that contain a limited supply of reagents, preventing over-amplification of certain regions. “There is a more even amplification, so you get better representation of the genome,” Adam Ameur, a bioinformatician at Uppsala University in Sweden, tells The Scientist.
By Holly Barker, PhD
Article can be accessed on: The Scientist