Researchers should now be able to study mutations in any human tissue due to a novel sequencing breakthrough by a team of scientists at the Wellcome Sanger Institute. The novel method makes it possible to more accurately investigate how genetic changes occur in human tissues.

Nanorate sequencing is extremely accurate and allows detection of new mutations in non-dividing cells, something that was difficult with earlier techniques. The sequencing method, also known as NanoSeq, uses duplex sequencing. This involves random molecular tagging of double-stranded DNA to improve accuracy and allow better detection of mutations.

Duplex sequencing is not new, but earlier iterations of the technique made it very difficult to detect mutations only present in single cells or small sections of non-dividing tissue. Mutations occur over time in our non-germline cells (at a rate of 15–40 per year) and these are often only in single or small groups of cells. While many such variants are harmless, some may cause cancer or other diseases later on.

This new NanoSeq technique took four years to develop and involved a careful refining of currently used duplex sequencing techniques. For example, using more specific enzymes to cut DNA more accurately and also better bioinformatics analysis of the sequencing data. It has an error rate of less than five errors per billion base pairs in single DNA molecules from cell populations. It also does not require invasive tissue biopsies and samples can be taken using skin or tissue swabs.

Robert Osborne, PhD, previously based at the Sanger, and Inigo Martincorena, PhD, a group leader at the Sanger led the research study “Somatic mutation landscapes at single-molecule resolution,” which is published in Nature.

“Somatic mutations drive the development of cancer and may contribute to aging and other diseases. Despite their importance, the difficulty of detecting mutations that are only present in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. Here, to overcome these limitations, we developed nanorate sequencing (NanoSeq), a duplex sequencing protocol with error rates of less than five errors per billion base pairs in single DNA molecules from cell populations. This rate is two orders of magnitude lower than typical somatic mutation loads, enabling the study of somatic mutations in any tissue independently of clonality,” write the investigators.

“We used this single-molecule sensitivity to study somatic mutations in non-dividing cells across several tissues, comparing stem cells to differentiated cells and studying mutagenesis in the absence of cell division. Differentiated cells in blood and colon displayed remarkably similar mutation loads and signatures to their corresponding stem cells, despite mature blood cells having undergone considerably more divisions.

“We then characterized the mutational landscape of post-mitotic neurons and polyclonal smooth muscle, confirming that neurons accumulate somatic mutations at a constant rate throughout life without cell division, with similar rates to mitotically active tissues.

“Together, our results suggest that mutational processes that are independent of cell division are important contributors to somatic mutagenesis.”

Technically challenging

“Detecting somatic mutations that are only present in one or a few cells is incredibly technically challenging,” said Osbourne, in a press statement. “You have to find a single letter change among tens of millions of DNA letters and previous sequencing methods were simply not accurate enough. Because NanoSeq makes only a few errors per billion DNA letters, we are now able to accurately study somatic mutations in any tissue.”

The team tested their method to evaluate levels of mutations in stem cells and non-dividing cells in the blood, neurons, and colon. They found similar levels of mutations in adult, differentiated blood and colon cells compared with stem cells in the same tissues. This was surprising for blood cells as they undergo frequent cell division. Neuronal cells, in contrast, seemed to gradually accumulate mutations over time.

“It is often assumed that cell division is the main factor in the occurrence of somatic mutations, with a greater number of divisions creating a greater number of mutations. But our analysis found that blood cells that had divided many times more than others featured the same rates and patterns of mutation. This changes how we think about mutagenesis and suggests that other biological mechanisms besides cell division are key,” noted Federico Abascal, PhD, a researcher in Martincorena’s group at the Sanger and the first author of the paper.

The researchers are now planning to develop and refine the technique further in more types of tissue and cell samples.

“The application of NanoSeq on a small scale in this study has already led us to reconsider what we thought we knew about mutagenesis, which is exciting,” said Martincorena. “NanoSeq will also make it easier, cheaper and less invasive to study somatic mutation on a much larger scale. Rather than analyzing biopsies from small numbers of patients and only being able to look at stem cells or tumor tissue, now we can study samples from hundreds of patients and observe somatic mutations in any tissue.”

The team believes that being able to more accurately detect mutations in single DNA molecules could transform the understanding of somatic mutagenesis and allow non-invasive studies on large-scale cohorts.

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