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

## TL;DR

A new method called CBTi-seq enables high-resolution, cost-effective analysis of full-length RNA in single cells, capturing detailed gene variations and splicing patterns.

## Contribution

CBTi-seq introduces a novel approach using Tn5 transposase and combinatorial barcodes to achieve high-resolution, low-cost single-cell transcriptome sequencing.

## Key findings

- CBTi-seq enables unambiguous reconstruction of splice variants and structural variations with base-pair precision.
- The method reduces sequencing costs and work time while improving sensitivity and resolution compared to existing methods.
- Cell-type-specific alternative splicing patterns are robustly identified in gene-edited and human testicular cells.

## 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.

CBTi‐seq is reported, leveraging Tn5 transposase‐mediated molecular assembly of combinatorial barcodes and UMIs for high‐resolution multiplexed sequencing of the full‐length transcriptome in single cells. This approach achieves molecular resolution by end‐to‐end sequencing, enabling reconstruction of splice variants and accurate quantification of transcript abundance. CBTi‐seq offers superior sensitivity and resolution, significantly reducing costs (≈0.7 USD cell−1) and fast work time (≈5 h).