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

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.