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Technological advancements in single-molecule DNA analysis have improved our understanding of the human genome, the microbiome, and the genetic basis of diseases. However, these methods traditionally require substantial amounts of DNA, limiting their application in scenarios where only small quantities are available, such as tumor biopsies.
Researchers at Gladstone Institutes have recently developed two innovative tools that dramatically reduce the amount of DNA needed for single-molecule analysis by 90 to 95 percent. Their findings, published in Nature Genetics, present promising new avenues for biological research that were previously unattainable.
Enhanced DNA Tagging for Comprehensive Analysis
One of the newly developed tools, "single-molecule real-time sequencing by tagmentation" (SMRT-Tag), builds upon existing protocols for mapping DNA base sequences and methylation patterns. Methylation, crucial for gene expression, is a key focus in understanding various diseases. Traditional methods struggle with small DNA samples because they require amplification, which can strip away methylation patterns and introduce errors.
“When we have very little DNA to work with, we can’t just make more copies of the DNA and apply our usual protocols,” explains Vijay Ramani, PhD, assistant investigator at Gladstone and senior author of the study. “Making copies would strip away these methylation patterns and introduce other errors.”
To overcome this, Ramani's team adapted a "tagmentation" process, which uses the bacterial protein Tn5 to simultaneously cut DNA into fragments and tag them for further analysis. Although tagmentation is typically used for short DNA fragments, the team optimized it for long fragments of about 3,000 to 5,000 base pairs. This involved extensive testing of various conditions with different buffers, enzymes, and temperatures to ensure reliability.
“It was quite a heroic effort by the staff and students in my lab,” Ramani says. “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 Insights from Minimal DNA Samples
Once optimized, SMRT-Tag demonstrated performance comparable to established protocols, but with significantly lower DNA requirements, approximately the amount found in as few as 10,000 cells. This breakthrough enables high-coverage sequencing with minimal samples.
“Using gold-standard single-molecule sequencing machinery, no one has ever sequenced such a small amount of DNA to the coverage we’ve now achieved,” Ramani states.
The team further enhanced their approach by combining SMRT-Tag with SAMOSA (single-molecule adenine methylated oligonucleosome sequencing assay), which they previously developed. SAMOSA-Tag, the new combined tool, enables the assessment of chromatin accessibility—a measure of how easily gene expression machinery can access DNA—using much less DNA than before.
In a practical demonstration, SAMOSA-Tag was applied to prostate cancer cells from both an initial tumor and a metastatic tumor transplanted and grown in mice. The tool revealed differences in chromatin accessibility that could indicate key drivers of cancer metastasis.
“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,” says Siva Kasinathan, M.D., Ph.D., who co-led the study with Ramani. “We think there’s some low-hanging fruit there that could unlock some new biology, which could be important for helping patients down the line.”
Collaborative Efforts and Future Directions
Ramani's team continues to refine SMRT-Tag and SAMOSA-Tag to work with even smaller DNA samples. They also actively share and update their protocols online, encouraging feedback and collaboration from the scientific community.
“The community and people involved are really important in the story of this work,” Ramani notes, highlighting the collaborative nature of the research.
In particular, Ramani acknowledges his long-standing collaboration with Kasinathan, a clinical fellow at Lucille Packard Children's Hospital at Stanford University and a visiting scientist at Gladstone. “It’s been so meaningful to work with one of my closest friends to publish what we think will be very impactful work for human health.”
Publication Details
Nanda, A.S., Wu, K., Irkliyenko, I. et al. Direct transposition of native DNA for sensitive multimodal single-molecule sequencing. Nat Genet (2024). https://doi.org/10.1038/s41588-024-01748-0