scLANTERN: High-Throughput Retrospective Lineage Tracing via Full-Length Single-Cell Transcriptomics and Expressed Repeat Variation

Understanding the lineage relationships among individual cells is a key pursuit of modern biology, essential for unraveling the complexities of developmental processes and the adaptive mechanisms of disease progression, particularly in oncology. Retrospective single-cell clonal tracing has emerged as a transformative approach, offering a unique window into the evolutionary trajectories of cancer within clinical samples. While short-read single-cell transcriptomics (scRNA-seq) has revolutionized our ability to map cell states across human tumor atlases, it remains fundamentally limited in its capacity to link these states with high-resolution genomic alterations and the evolutionary trajectories inferred from these natural variants. Integrating somatic mutation discovery with transcriptomic profiles at single-cell resolution often requires separate, costly, and low-throughput genomic assays. Furthermore, existing methods frequently rely on exogenous genetic labeling or are restricted to short-read sequencing, which typically fails to resolve complex genomic rearrangements, large indels, or variations within highly repetitive regions—such as short tandem repeats (STRs)—that could serve as potent endogenous clonal markers.

To address these limitations, we present scLANTERN (single cell Long-reAd liNeage Tracing via Expressed Repeat variatioN) , a novel, high-throughput methodology for the simultaneous discovery of highly mutable somatic variants and cell-state annotation from full-length single-cell transcriptomes. Utilizing Multiplexed Arrays for Sequencing (MAS-Seq) on the PacBio Revio platform, scLANTERN bypasses the resolution constraints of short-read technologies. The process begins with the efficient isolation of individual cells or nuclei and concludes with the generation of clonal/lineage phylogenetic trees, integrated with detailed annotations of gene isoforms and variants. scLANTERN can be deployed after running canonical short read single-cell profiling with the remaining cDNAs, allowing for data-driven selection of optimal samples. Importantly, this technology can be deployed on primary tissue samples after canonical single cell/nuclei profiling and does not require exogenous genetic barcoding, therefore enabling the establishment of high-resolution lineage tracing of primary tissues during normal development or tumor evolution.