Researchers Advance Single-Molecule DNA Sequencing Techniques

In recent years, technologies that allow scientists to investigate a person's DNA at single-molecule resolution have greatly increased the knowledge of the human genome, microbiome, and the genetic basis of disease. With such a precise picture of DNA, genetic variations and structural features that were previously undetected using other sequencing technologies can be seen.

However, today's gold-standard technologies for single-molecule analysis often require at least 150,000 human cells containing millions of individual DNA molecules.

This implies that researchers cannot use these methods when only a few thousand cells are accessible, as is the case in many tumor biopsies, limiting the technologies' scientific and therapeutic promise.

Gladstone Institutes researchers have created two novel technologies for single-molecule analysis that reduce the quantity of DNA required by 90-95 percent.

Their research, published in the journal Nature Genetics, demonstrates how these techniques might help scientists answer biological issues that were previously impossible to answer.

We have been working toward creating these methods for a very long time, we are really excited to see what discoveries will now be possible.”

Vijay Ramani, PhD, Study Senior Author and Assistant Investigator, Gladstone Institutes

‘Tagging’ DNA for a Clearer View

The first of the new tools, known as "single-molecule real-time sequencing by tagmentation," or SMRT-Tag, expands on established protocols for mapping the sequence of bases in a long DNA fragment while also mapping the locations of chemical structures known as methyl groups that run along the length of the DNA.

Methyl groups have a vital role in gene expression and are, therefore, necessary for understanding disease; hence, it is critical to understand how they are structured in DNA.

Ramani added, “When we have very little DNA to work with, we cannot just make more copies of the DNA and apply our usual protocols, making copies would strip away these methylation patterns and introduce other errors.”

Instead, his team used a technique known as "tagmentation," which repurposes the bacterial enzyme Tn5 to break DNA molecules into smaller fragments while also labeling them with chemical components required for further research.

Actionable Data from Small Samples

Tagmentation is already used to sequence tiny segments of DNA when only small quantities of DNA are available; however, short snippets provide only limited information.

Ramani's team faced the task of perfecting the biochemistry of tagmentation to break up small quantities of DNA into lengthy pieces of 3,000 to 5,000 base pairs. Their technique "tags" the ends of each fragment with hairpin-shaped structures, resulting in large loops of DNA that can be reliably read by sequencer equipment.

Ramani stated, “It was quite a heroic effort by the staff and students in my lab. We had to test different versions of Tn5 and nearly 100 different conditions with different buffers, enzymes, and temperatures. When you’re working with such small amounts of DNA, any issue that causes any DNA loss is that much more of a problem.”

Actionable Data from Small Samples

After optimizing SMRT-Tag, the scientists proved that it operates as well as existing techniques while requiring far less DNA—about the amount found in as few as 10,000 cells.

Ramani further added, “Using gold-standard, single-molecule sequencing machinery, no one has ever sequenced such a small amount of DNA to the coverage we have now achieved.”

Next, his team paired SMRT-Tag with a technology they previously invented called SAMOSA, which stands for "single-molecule adenine methylated oligonucleosome sequencing assay."

SAMOSA reveals additional methylation patterns that represent chromatin accessibility—i.e., how easily gene expression machinery can reach distinct segments of DNA.

With the novel SAMOSA-Tag method, the researchers were able to measure chromatin accessibility with far less DNA than previously required. To demonstrate, researchers implanted and developed prostate cancer cells in mice, some from the patient’s initial tumor and others from a tumor that had migrated to a different region in the body. The technique found disparities in chromatin accessibility, which point to potential important causes of cancer spread.

This is just one example of how our tools could be applied to clinically relevant samples in cancer and other diseases where DNA is in short supply, we think there is some low-hanging fruit there that could unlock some new biology, which could be important for helping patients down the line.”

Siva Kasinathan, MD, PhD, Study Co-Lead Author, Gladstone Institutes

Ramani's team is now improving SMRT-Tag and SAMOSA-Tag to function with even smaller quantities of DNA. They also continue to share and update their procedures online, encouraging feedback and cooperation from other academics.

Ramani noted, “The community and people involved are really important in the story of this work.”

He concluded, “It is been so meaningful to work with one of my closest friends to publish what we think will be very impactful work for human health.”