Nanorate sequencing allows researchers to identify changes to individual genetic sequencing letters among millions of DNA letters contained in a single cell

Detecting genetic mutations in cells requires genomic sequencing that, until now, has not been accurate enough to spot minute changes in DNA sequences. Many clinical laboratory scientists know this restricted the ability of genetic scientists to identify cancerous mutations early in individual cells.

Now, researchers at the Wellcome Sanger Institute in the United Kingdom have developed a new method of genetic sequencing that “makes it possible to more accurately investigate how genetic changes occur in human tissues,” according to Genetic Engineering and Biotechnology News (GEN).

This development suggests a new, more sensitive tool may soon be available for anatomic pathologists to speed evaluation of pre-cancerous and cancerous tissues, thereby achieving earlier detection of disease and clinical intervention.

Called Nanorate Sequencing (NanoSeq for short), the new technology enables researchers to detect genetic changes in any human tissues “with unprecedented accuracy,” according to a news release.

The Wellcome Sanger Institute researchers published their findings in the journal Nature, titled, “Somatic Mutation Landscapes at Single-Molecule Resolution.”

How Somatic Mutations Drive Cancer, Aging, and Other Diseases

NanoSeq enables the detection of new mutations in most human cells—the non-dividing cells—GEN explained, calling Wellcome Sangar Institute’s new technology a “breakthrough” in the use of duplex sequencing.

Until now, genomic sequencing has not been “accurate enough” for this level of detection, Sanger stated in the news release. Thus, there was little opportunity to enhance exploration of new mutations in the majority of human cells.

Further, the findings of the Sanger study suggest that cell division may not be the primary cause of somatic mutations (changes in the DNA sequence of a biological cell).

In their paper, the researchers discussed the importance of somatic mutations. “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,” the researchers wrote in Nature.

Refining Duplex Sequencing and Improving PCR Testing

In their study, Sanger researchers assessed duplex sequencing and found errors concentrated at DNA fragment ends. To them, this suggested “flaws” in preparation for DNA sequencing.

Duplex sequencing is an established technique “which sequences both strands of a DNA molecule to remove sequencing and polymerase chain reaction (PCR) errors,” explained a Science Advisory Board article.

Robert Osborne, PhD

“Detecting somatic mutations that are only present in one or a few cells is incredibly technically challenging. You have to find a single letter change among tens of millions of DNA letters and previous sequencing methods were simply not accurate enough,” said Robert Osborne, PhD (above), former Principal Staff Scientist at Sanger who led development of NanoSeq, in the news release. Osborne is now COO of Biofidelity, a cancer diagnostics developer in Cambridge, United Kingdom. This research may eventually give clinical laboratories and surgical pathologists useful new tools that enable earlier, more accurate diagnosis of cancer. (Photo copyright: Cambridge Independent.)

Re-evaluating Mutagenesis and Cell Division with NanoSeq

It took the Sanger researchers four years to create NanoSeq. They “carefully refined” duplex sequencing methods using more specific enzymes to aid DNA cutting and bioinformatics analysis, Clinical OMICS noted.

Then, they put NanoSeq’s sensitivity to the test. They wanted to know if its low error rate meant that NanoSeq could enable study of somatic mutations in any tissue. This would be important, they noted, because genetic mutations naturally occur in cells in a range of 15 to 40 mutations per year with some changes leading to cancer.

The scientists compared the rate and pattern of mutation in both stem cells (renewing cells supplying non-dividing cells) and non-dividing cells (the majority of cells) in blood, colon, brain, and muscle tissues.

The Sanger study found:

  • Mutations in slowly dividing stem cells are on track with progenitor cells, which are more rapidly dividing cells.
  • Cell division may not be the “dominant process causing mutations in blood cells.”
  • Analysis of non-dividing neurons and rarely-dividing muscle cells found “mutations accumulate throughout life in cells without cell division and at a similar pace” to blood cells.

“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 divisions are key,” said Federico Abascal, PhD, First Author and Sanger Postdoctoral Fellow, in the news release.

Using NanoSeq to Scale Up Somatic Mutation Analyses

“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,” said Inigo Martincorena, PhD, Senior Author and Sanger Group Leader, in the news release.

More research is needed before NanoSeq finds its way to diagnosing cancer by anatomic pathology groups. Still, for diagnostics professionals and clinical laboratory leaders, NanoSeq is an interesting development. It appears to be a way for scientists to see genetic changes in single cells and mutations in a handful of cells that evolve into cancerous tumors, as compared to those that do not.

The Sanger scientists plan to pursue larger follow-up NanoSeq studies.

—Donna Marie Pocius

Related Information:

NANOSEQ: Nanorate Sequencing, Ultra-Accurate Detection of Somatic Mutations

Major Advance Enables Study of Genetic Mutations in Any Tissue

NanoSeq Technique Improved to Detect New Non-Dividing Cell Mutations

Somatic Mutation Landscape at Single-Molecule Resolution

New Method Allows for Study of Genetic Changes in Individual DNA Molecules

Sanger Institute Improves NanoSeq Method to Detect New Mutations in Non-Dividing Cells

Wellcome Sanger Institute’s NanoSeq Sequencing Breakthrough Enables Study of DNA Mutations from Any Human Tissue