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Adding spatial resolution and technical improvements to a novel single-cell DNA methylation sequencing technology

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NHGRI - National Human Genome Research Institute

ABSTRACT DNA methylation (DNAme) is a core layer of epigenetic regulation with central roles in the establishment and maintenance of cellular identity. Methods to profile DNAme at single-cell resolution are needed to elucidate epigenetic networks governing cell state in healthy tissues and understand their dysregulation in disease and aging. However, existing methods for single-cell methylome profiling are highly inaccessible, and no methods to experimentally profile both DNAme and spatial location currently exist. I have developed a novel method for single-cell DNAme profiling that leverages the widely available 10x Single Cell Multiome and NEB EM-seq kits, which I have named droplet-implemented single-cell DNA methylation sequencing (discDNAme). Applied to nuclei isolated from mouse brain, discDNAme recovered ~3,000 high-quality methylomes that clustered into clearly separated neuronal and non-neuronal subtypes and displayed stereotyped patterns of CpG dinucleotide methylation around key genomic features. However, these measurements lack spatial information on the native tissue contexts of profiled cells, and the protocol’s per-cell library size is lower than current gold-standard methods for measuring single-cell DNAme. I propose (1) development of a spatially resolved single-cell DNA methylation technology by combining discDNAme with slide-tags, a platform for spatially resolved single-nucleus RNA- and/or ATAC-seq developed by our group. To integrate slide-tags with discDNAme, I will develop a protocol in which the “spatial barcode oligos” we use to position nuclei are physically separated from genomic DNA prior to unmethylated cytosine conversion, benchmark this technology in the mouse hippocampus, and apply it to study glioblastoma multiforme. I further propose (2) experiments to improve and benchmark my discDNAme technology. I will systematically test various independent approaches to increase library complexity at different steps of the discDNAme protocol, combine these optimizations into a second-generation protocol, and benchmark this against our original protocol and other leading methods for single-cell DNAme profiling. Completion of this proposal will result in (1) the first high-resolution method to measure spatially resolved single- cell methylomes—a major advancement in spatial omics technologies—and (2) an accessible yet capable tool for single-cell DNAme profiling that will open single-cell DNAme studies to the broader single-cell community.

Up to $44K
2028-01-31
health research
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Addressing disparities in pediatric intestinal failure

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NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

The proposed K23 Career Development Award will enable Vikram Raghu, MD, MS, to establish an independent research career focused on improving outcomes in pediatric intestinal failure. Pediatric intestinal failure is a rare, chronic, life-altering condition that requires many children to depend on daily infusions of parenteral nutrition to survive. Complications include life-threatening sepsis events, progressive liver disease, and venous thrombosis, with mortality estimated at over 10% by six years. Despite advances in care, outcomes vary widely, and emerging evidence suggests that geographic and socioeconomic factors strongly influence survival, healthcare use, and quality of life in this population. To address this knowledge gap, Dr. Raghu has assembled a multidisciplinary team of mentors and designed a training program in health disparities research (with a focus on socioeconomic and geographic drivers), multicenter health utility measurement, patient preference elicitation, transplant policy, and leadership in research conduct. These skills will support his overarching goal of optimizing intestinal failure management through decision sciences. The central hypothesis of this project is that variation in outcomes is partly explained by differences in geographic and socioeconomic conditions, such as access to healthcare, school resources, and environmental context, independent of clinical severity. To evaluate this hypothesis, he will accomplish the following specific aims: (1) Identify the effect of the Child Opportunity Index on survival and healthcare utilization in pediatric intestinal failure; (2) Examine socioeconomic and geographic differences in quality of life among children with intestinal failure using the Child Health Utility 9D; and (3) Determine patient and caregiver priorities for addressing socioeconomic and geographic barriers to care through a modified Delphi process and discrete choice experiment. By successfully completing these training objectives and research aims, Dr. Raghu will be positioned to become an independent investigator with the expertise to integrate decision modeling, disparities research, and patient-centered methods to improve outcomes for children with intestinal failure.

Up to $177K
2031-01-31
health research