Finally, scientists sequence single cells with long-read technology

JTraditional sequencing is often likened to making a smoothie: researchers mix together a bunch of cells, get an average sequence, and draw conclusions about the ingredients that make up the mush. More recently, scientists have gained the ability to perform single-cell sequencing, which can reveal rare variations between cells and the evolution of cell lines. But current methods require reading the genome in short sections and therefore often fail to capture complex repeating regions, which scientists increasingly associate with health and health. disease. Long-read technologies could overcome this pitfall; however, their methods require far more DNA than can be extracted from a single cell. Long read single cell 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 applied the long-read technology to single cells, says Alexander Hoischen, a genomics technology researcher at Radboud University Medical Center in the Netherlands who was not involved in the research. “It was unthinkable just two or three years ago,” says Hoischen.

The exploit may allow a more detailed examination of the mutations underlying all kinds of diseases, experts say The scientist.

See “Infographic: The Sequencing and Assembly of the Human Genome”

It was unthinkable just two or three years ago.

—Alexander Hoischen, Radboud University Medical Center

Over the past decade, reads — the product of DNA sequencing — have gotten 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 difficult-to-map duplicate regions on a chromosome.

However, long-read sequencing requires a ton of DNA. Several micrograms of genetic material are needed, but “a single cell only contains six picograms”, explains Joanna Hard, a cell biologist at ETH Zurich in Switzerland. “Substantial amplification is therefore required before it can be sequenced” using long-read methods, she says.

And that’s where things get tricky, says Hård, because the main methods used to amplify DNA are subject to “amplification bias”: the tendency for some sequences to speed up at the expense of others. Now, Hård and his colleagues have obtained long reads from single cells using an improved DNA amplification method. Although not yet peer-reviewed, the findings were reported in a preprint uploaded to bioRxiv January 23.

See “Major scientific collaboration aims to complete the human genome”

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 amount of reagents, preventing certain regions from being overamplified. “There’s more uniform amplification, so you get a better representation of the genome,” Adam Ameurbioinformatician at Uppsala University in Sweden, says The scientist.

The researchers performed the droplet-based amplification on individual human T cells and then generated long reads using PacBio HiFi Technology. Compared to short-read sequencing, the new method captured four times more structural variants— large DNA rearrangements — including those located in inaccessible “dark regions” of the genome.

The researchers used the new approach to sequence the DNA of two different T cells obtained from the same person. Their sequencing data revealed 28 somatic mutations that distinguished the two cells, including mitochondrial DNA changes. The new method could allow scientists to study the impact of somatic mutations in a myriad of diseases.

For example, tumors often exhibit a mosaic of genetic variation, as different cells independently acquire mutations, called subclonal mutations, that make the tumor more aggressive. Improved single-cell DNA sequencing could help ‘uncover subclonal mutations that often lurk in cancer,’ says Christopher Masona biophysicist from Weill Cornell Medicine in New York who was not involved in the study.

See “2D genetic map of prostate cells shows cancer growth”

Healthy cells also accumulate mutations throughout their lives, although this is less alarming in skin and gut cells, which are regularly replenished, than in our long-lived brain cells. But somatic mutations in neurons and other brain cells could impact brain function and contribute to neurological disorders, including schizophrenia, Tourette’s disease and autism.

Constantine Theofanopoulou, a Hunter College researcher who was not involved in the study, says her research has already benefited from advances in DNA sequencing. Long reads of vertebrate genomes helped her discover the evolutionary history receptors and ligands that enable social communication. But long reads obtained from single cells could be a crucial tool for identifying genetic variants in the speech disorders she studies, she says.

However, the technique is not perfect, admit the researchers. Although better than current DNA scaling methods, droplet-based amplification can produce errors and chimeras, where non-neighboring parts of the genome are stuck together. Although they were able to identify and remove the chimeras in the current works, their strict filtering method also rejected the correct reads. The team is working on optimizing conditions to reduce errors during amplification.

“I see this as a proof-of-principle study, where we show that everything that has been done with long reads in large samples can also be done at the single-cell level,” Ameur says. “Now we can really study individual cells in more detail,” he adds.

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