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Author ORCID Identifier
https://orcid.org/0000-0002-6447-6433
Date Available
4-16-2026
Year of Publication
2026
Document Type
Doctoral Dissertation
Degree Name
Doctor of Philosophy (PhD)
College
Medicine
Department/School/Program
Neuroscience
Faculty
Mark T.W. Ebbert
Faculty
Richard C Grondin
Abstract
Long-read single-cell RNA sequencing provides an opportunity to understand human health and disease at isoform resolution, revealing cellular diversity and disease mechanisms difficult to resolve with bulk or short-read methodologies.
Using a modified PIPseq workflow and computational pipeline adapted for Oxford Nanopore (ONT) sequencing, we profiled isoform usage across immune cells, integrating marker expression and isoform discovery, generating the largest long-read single-cell dataset of human immune cells from a single individual to date. We identified non-canonical protein-coding variants of GZMB and CD3G enriched in unexpected cell types. We also discovered novel transcripts from CMC1 and LYAR with cell-type-specific signatures that were the predominant transcript within each gene, along with 126 novel isoforms from known and new genes.
Using the marker isoforms as a positive control, we developed a centroid-based computational framework that contextualizes novel genes and isoforms relative to reference genes with similar cell-type expression patterns. This enables functional inference of isoform divergence and novelty according to their biologically meaningful neighbors, rather than relying solely on host-gene annotation or similarity to known protein domains.
This work demonstrates the power of the technique for mapping the isoform landscape—the isonome—across tissues and disease contexts.
Digital Object Identifier (DOI)
https://doi.org/10.13023/etd.2026.106
Archival?
Archival
Funding Information
This work was supported by the National Institutes of Health [5T32AG078110-02 to University of Kentucky Sanders-Brown Center on Aging, AG078110 to Doyle] from 2023-2025. It was also supported by the National institutes of Health [R01AG068331 to Ebbert; GM138626 to Ebbert], the Alzheimer’s Association [2019-AARG-644082 to Ebbert], and the BrightFocus Foundation [A2020161S to Ebbert] from 2022 to 2023.
Recommended Citation
Doyle, Patricia Hayes, "A FRAMEWORK FOR CHARACTERIZING THE PERIPHERAL IMMUNE ISONOME USING LONG-READ SINGLE-CELL RNA SEQUENCING AND ITS RELEVANCE TO NEUROLOGICAL DISEASE" (2026). Theses and Dissertations--Neuroscience. 42.
https://uknowledge.uky.edu/neurobio_etds/42
Supplemental File for dissertation
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