Doyle, P. H.; Page, M. L.; Brandon, J. A.; Heberle, B. A.; White, B. J.; Stowe, A. M.; Ebbert, M. T. W.

Long-read single-cell RNA sequencing provides an opportunity to understand human health and disease at a new level that is difficult to resolve with either bulk or short-read methods. Specifically, with long-read single-cell sequencing, it is possible to investigate cellular diversity and disease mechanisms on the isoform level and define cell types at the RNA isoform level (i.e., marker isoforms) rather than solely at the gene level (i.e., marker genes). 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 profiles isoform usage among immune cell marker genes in primary PBMCs and incorporates isoform discovery. We identified 128 previously unannotated genes from known and new genes, several with distinct cell-type-specific patterns across immune cell types, and characterized the expression of marker gene isoforms across cell-types and T cell subtypes. We identified non-canonical protein-coding variants of GZMB and CD3G enriched in unexpected cell-types, including megakaryocytes and monocyte-derived cells. We also discovered novel transcripts from CMC1 and LYAR with cell-type-specific signatures that were the most predominantly expressed transcript within the gene. This study expands the versatility of long-read single-cell studies to not just relay what changes in isoform signatures were found, but to position these results within the functional context of the biology they impact. These results demonstrate the power of long-read single-cell sequencing as an approach to mapping the isoform landscape--the isonome--for transcriptomic profiling across tissues and disease contexts.