Existing genome-wide sequencing methods cannot provide base-pair level resolution and spatial location of a sequence at the same time. A new method combining sequencing and micro-imaging, developed by MIT scientists, called in situ genome sequencing, sequences DNA directly within intact biological samples and pinpoints where specific DNA sequences are located inside intact cells.

“How structure yields function is one of the core themes of biology,” said Edward S. Boyden, PhD, professor in neurotechnology, biological engineering, and brain and cognitive sciences at MIT’s McGovern Institute for Brain Research, and founder of Expansion Technologies. “And the history of biology tells us that when you can actually see something, you can make lots of advances.”

In unleashing the ability to directly visualize how an organism’s genome is packed inside its cells, this technology now makes it possible to ask questions about how different cell types interpret the genetic code, how DNA’s three-dimensional organization affects its function, and how structural changes and chromosomal rearrangements are associated with aging, cancer, brain disorders, and other diseases. Additionally, the authors noted, the new method also makes it possible to directly see how the genome interacts with regulatory factors.

“Some advances on the technology side have taken this from impossible to do to being possible,” said Fei Chen, PhD, core institute member at the Broad Institute of MIT and Harvard, and assistant professor in the department of stem cell and regenerative biology at Harvard University.

This new method builds on work in Boyden and Chen’s laboratories focused on sequencing RNA inside cells. In collaboration with Boyden and Chen, Jason D. Buenrostro, PhD, associate member of the Broad Institute, and an assistant professor in the department of stem cell and regenerative biology at Harvard University, attempted to adapt the technique for use with DNA.

“It was clear the technology they had developed would be an extraordinary opportunity to have a new perspective on cells’ genomes,” Boyden said.

In situ genome sequencing uses well-established protocols optimized for DNA fluorescence in situ hybridization (DNA FISH), to fix cells onto a glass surface to preserve their structure. Then the authors used a transposase enzyme to insert DNA sequencing adaptors into the fixed DNA, preserving genomic fragments in their native spatial organization. These genomic fragments are then circularized and amplified to generate a DNA sequencing library with thousands of spatially localized genomic fragments per nucleus. Finally, all the cells’ DNA is sequenced, and the location-identified short sequences are identified within longer segments pinpointing each fragment’s position.

Boyden, Buenrostro, and Chen, who began their collaboration several years ago, say the new technology represents a heroic effort on the part of MIT and Harvard graduate students Andrew Payne, Zachary Chiang, and Paul Reginato, who took the lead in developing and integrating its many technical steps and computational analyses. This involved introducing significant innovations in commercial sequencing methods.

The authors used this novel technology to visualize a genome as it reorganizes itself in early mouse embryos. Brightly colored representations of DNA sequenced inside a mouse embryo show how genetic information inherited from each parent remains compartmentalized immediately after fertilization, then gradually intertwines as development progresses. This new sequencing method reveals how patterns of genome organization, which very early in life vary from cell to cell, are passed on as cells divide, generating a memory of each cell’s lineage. Being able to watch these processes unfold across entire cells instead of piecing them together through less direct means offers a dramatic new view of development, the researchers said.

The team continues to improve the spatial resolution of the technique and adapt it to a broader range of cell types. They have made the method and associated software freely available to other labs. The researchers hope this new approach to DNA sequencing will change the way people think about studying the structure of the genome and will help illuminate patterns and consequences of genome organization across a variety of contexts.

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