Ebbert_lab_PBMC_long_read_single_cell_project

Long-read single-cell RNA sequencing provides an opportunity to understand human health and disease at a level difficult to resolve with bulk or short-read methods. This approach enables isoform-level investigation of cellular diversity and disease mechanisms and definition of cell-types, rather than using genes alone. Using a modified, microfluidic-free PIPseq workflow and computational pipeline adapted for Oxford Nanopore long-read sequencing, we generated the largest long-read single-cell dataset of human peripheral blood mononuclear cells (PBMCs) to date. This study profiled isoform usage across immune cells, integrating marker expression and isoform discovery. We identified 128 novel isoforms from known and new genes, several with distinct cell-type-specific patterns, and characterized marker gene isoform expression across cell-types. Non-canonical protein-coding variants of GZMB and CD3G were enriched in unexpected cell-types, including megakaryocytes and monocyte-derived populations. We also discovered novel transcripts from CMC1 and LYAR with cell-type-specific signatures that were the predominantly expressed transcript within the gene. This study expands versatility of long-read single-cell studies to not only relay changes in isoform signatures, but to position them within the functional context of the biology they impact. These results demonstrate the power of long-read single-cell sequencing for mapping the isoform landscape—the isonome—across tissues and disease contexts. University of Kentucky Sanders-Brown Center on Aging, AG078110 to Doyle; R01AG068331 to Ebbert; GM138626 to Ebbert; NS088555 to Stowe]; Alzheimer’s Association [2019-AARG-644082 to Ebbert]; the Bright Focus Foundation [A2020161S to Ebbert]; and PhRMA Foundation Predoctoral Drug Discovery Fellowship to Heberle.