High‐Resolution Multiplexed Sequencing of Single‐Cell Full‐length Transcriptome Via Combinational Barcoded Tn5 Transposon Insertion

Abstract The technological advancements in single‐cell transcriptome analysis make significant progress in both depth and breadth. However, balancing the cell analysis throughput with full‐length transcript coverage remains a persistent challenge. Here, CBTi‐seq (Combinational Barcoded Tn5 Transposon Insertion sequencing) is reported, leveraging Tn5 transposase‐mediated molecular assembly of combinatorial barcodes and unique molecular identifiers (UMIs) to enable high‐resolution multiplexed sequencing of the full‐length transcriptome in single cells. This approach achieves molecular resolution by end‐to‐end sequencing, enabling unambiguous reconstruction of splice variants and structural variations with base‐pair precision. The design of orthogonal combination barcode Tn5 reduces DNA barcode diversity while enhancing multiplexing flexibility, and Tn5‐delivered UMIs insertion eliminates read bias, providing accurately quantifies transcript abundance through the tagging of each fragment. The method is compatible with both single‐cell and spatially resolved tissue microenvironment. Compared with commercial terminal library and other full‐length sequencing methods, CBTi‐seq achieves superior sensitivity and resolution while significantly reducing costs and work time (≈5 h). Moreover, cell‐type‐specific alternative splicing patterns are robustly identified in both gene‐edited cells and human testicular cells, leveraging this high‐resolution capability to further reveal modality dynamic events and isoform switching independent of gene expression changes during spermatogenesis with the potential to reproductive development and diagnostic treatment.