Enhancing DNA Analysis: Long-Read Technology for Single-Cell Insights into Genetic Variation

Researchers from SciLifeLab National Genomics Infrastructure (NGI), Karolinska Institutet and Uppsala University have achieved a new advancement in DNA analysis, combining DNA amplification with the latest sequencing techniques to apply long-read technology to individual cells. This innovation holds the potential to provide a more comprehensive insight into disease-associated mutations.

In recent years, long-read sequencing technology has led to remarkable achievements, including the final completion of a human reference genome, and discovery of new genetic variation outside the detection range of short-read technologies. A future milestone is the reconstruction of complete genome sequences from individual cells. However, due to technical limitations, this is extremely challenging.

“Long-read sequencing requires a substantial DNA quantity, typically several micrograms, while a single cell contains merely six picograms”, as emphasized by Joanna Hård, postdoc at ETH Zurich in Switzerland. This implies that significant amplification of DNA is needed before conducting long-read sequencing on single cells.

To minimize amplification bias, the team developed 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. “The amplification in droplets gives a more uniform amplification, so you get better representation of the genome”, says Adam Ameur, associate professor and bioinformatician at SciLifeLab, NGI, Uppsala university. 

The project came about as a collaboration between researchers at SciLifeLab NGI, the Danish company Samplix and Karolinska Institutet. “Together we had access to the necessary samples, equipment and know-how to attempt to analyze DNA from individual cells with long-read sequencing. The success of this project shows that SciLifeLab NGI can play an important role to develop new applications in genomics” says Ameur.

The researchers used the new method to perform PacBio sequencing of DNA from two different T-cell clones originating from the same person. This revealed all types of genetic variation, including structural variation and repetitive sequences, which had previously not been possible to study at the single cell level. Since the technique can detect genetic differences in individual cells, it has the potential for studying somatic structural variation in cells affected by disease, including for example cancer and neurological disease

However, it’s important to note that the technique has its imperfections, as acknowledged by the researchers. Although an improvement over existing DNA scaling methods, droplet-based amplification can introduce errors and chimeras, where non-adjacent genome segments are mistakenly joined together. While the team successfully identified and removed chimeras in their current work, their stringent filtering approach also excluded correct reads. The researchers are actively working on optimizing conditions to minimize errors during amplification.

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

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Last updated: 2023-09-06

Content Responsible: Hampus Persson(