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

Sep 29, 2026

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Single-cell transcriptome analysis has advanced in both depth and breadth, yet reconciling high cellular throughput with true full-length transcript coverage remains difficult. CBTi-seq (Combinational Barcoded Tn5 Transposon Insertion sequencing) uses Tn5 transposase to assemble combinatorial barcodes and UMIs for high-resolution multiplexed full-length sequencing. End-to-end reads enable base-pair-precision reconstruction of splice variants and structural variation, while orthogonal barcoded Tn5 reduces barcode diversity, improving multiplexing flexibility and quantifying transcript abundance without read bias. The workflow supports single-cell and spatial tissue contexts, offering higher sensitivity and resolution with ~5 h runtime, and enables robust isoform switching analysis during spermatogenesis.

LanguageEnglish

CategoryResearch & Report

FormatPDF

Pages14

Reading Time35 min

AccessFree to download

Introduction

Single-cell RNA sequencing and throughput–coverage trade-offs

Limitations of end-bias and short-read full-length capture

Rationale for high-resolution full-length strategies

RESEARCH ARTICLE [www.advancedscience.com](www.advancedscience.com) High-Resolution Multiplexed Sequencing of Single-Cell Full-length Transcriptome Via Combinational Barcoded Tn5 Transposon Insertion Liyong He, Kaitong Dang, Qian Sun, Wenjia Wang, Wenbo Li, Wenyi Zhang, Kaiqiang Ye, Handong Wang, Zhengyue Li, Yan Guo, Zheng Li, Chencheng Yao, Peng Li, Yan Huang, and Xiangwei Zhao* 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. 1. Introduction Single-cell RNA sequencing (scRNA-seq) has provided a powerful tool in biology research to reveal the heterogeneity of complex samples and discover novel cell types in the past decade. [1–5] Continued advances in molecular barcoding techniques and integration of droplet microﬂuidic-/microwell array-based platforms allow gene expression proﬁling across thousands to millions of single cells. [6–10] However, most high-throughput scRNA-seq methods rely on barcoded oligo-dT primers to capture and initiate reverse transcription (RT) from the poly(A) tail of mRNAs and convert the barcode sequences to the RNA end. These methods introduce inherent technical limitations, only detect a short part of the RNA (≈400–600 base pairs) adjacent to the end of the transcripts, resulting in obvious terminal bias. Although such bias won’t perturb the result of proﬁling diﬀerential gene expression in high numbers of cells, the limited coverage of transcripts makes it diﬃcult to reveal structure-related information, such as alternative splicing (AS) events and allelic variants. [11–13] L. He, K. Dang, W. Wang, W. Zhang, K. Ye, H. Wang, Y. Huang, X. Zhao State Key Laboratory of Digital Medical Engineering School of Biological Science & Medical Engineering Southeast University Nanjing 211189, China E-mail: [xwzhao@seu.edu.cn](xwzhao@seu.edu.cn) The ORCID identiﬁcation number(s) for the author(s) of this article can be found under [https://doi.org/10.1002/advs.202516013](https://doi.org/10.1002/advs.202516013)[ ](https://doi.org/10.1002/advs.202516013)© 2025 The Author(s). Advanced Science published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. DOI: 10.1002/advs.202516013 Q. Sun, Z. Li State Key Laboratory of Reproductive Medicine and Oﬀs

FAQs

What problem does CBTi-seq target in single-cell RNA sequencing?

It targets the persistent trade-off between analyzing many cells and achieving full-length transcript coverage without strong terminal bias.

How does CBTi-seq achieve molecular-resolution sequencing of full-length transcripts?

It combines combinatorial barcoded Tn5 transposon insertion with UMIs, enabling end-to-end sequencing that reconstructs splice variants and structural variations with base-pair precision.

What improvements does the paper report compared with commercial terminal library and other full-length methods?

CBTi-seq reports superior sensitivity and resolution while significantly reducing costs and work time to approximately 5 hours.

Sep 29, 2026

Upload by Sarah