NanoGAM: a method of studying the three-dimensional structure of the genome, through the use of long read DNA sequencing of ultracryosectioned nuclear profiles by laser microdissection
DNA is packaged inside the cell nucleus in a hierarchical fashion from nucleosomes up to the level of whole chromosomes. Those are highly orchestrated 3D conformations that adapt during development, in response to stimuli, or in disease. The development of the Hi-C method was a breakthrough in understanding the three-dimensional structure of the entire genome. However, this method has important limitations, due to their reliance on digestion and ligation to capture interacting DNA segments. GAM is an alternative method, it does not use ligation, and the 3D distances are measured by combining ultracryosectioning with laser microdissection and DNA sequencing. Structurally preserved, fixed cells embedded in sucrose and frozen are thinly cryosectioned, before isolating single nuclear profiles by laser microdissection. The DNA content of each nuclear profile is extracted, amplified and sequenced. Loci that are closer to each other in the nuclear space (but not necessarily on the linear genome) are detected in the same nuclear profile more often than distant loci. GAM may have clinical application because cells can be fixed in tissue and only hundreds, not millions, of cells are needed to create a contact map. This allows the study of the threedimensional structure of the genome in pathological tissues. While working with GAM data, I identified genome regions that are poorly represented in the data, often in regions close to centromeres or telomeres. They are excluded from the analysis. Such regions with repeats are poorly identifiable during Illumina sequencing, where short reads are obtained. Therefore, their role in the three-dimensional structure of the genome is unknown. I want to improve the GAM method by using Oxford Nanopore long read sequencing. I will use F123-CASTx129 mouse embryonic cell line. For this strain, a lot of single mutations distinguishing individual alleles are known, which allows to distinguish information about the threedimensional structure for homologous chromosomes. I plan to analyze the sequence-structure relationship in phased chromosomes and create a model of three-dimensional structure for all chromosomes in the diploid cell nucleus.
As part of the BIOTECHMED-2_Start Research Grant “Excellence Initiative – Research University” Research Area – Biotechnology and Biomedical Engineering, WUT
Warsaw University of Technology, Centre for Advanced Materials and Technologies CEZAMAT
Faculty of Mathematics and Information Sciences, Warsaw University of Technology
119 994 pln
Teresa Szczepińska, PhD Eng.