• High-Resolution Multiplexed Se...

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

Title:
High-Resolution Multiplexed Sequencing of Single-Cell Full-length Transcriptome Via Combinational Barcoded Tn5 Transposon Insertion.
Authors:
He L; State Key Laboratory of Digital Medical Engineering, School of Biological Science & Medical Engineering, Southeast University, Nanjing, 211189, China., Dang K; State Key Laboratory of Digital Medical Engineering, School of Biological Science & Medical Engineering, Southeast University, Nanjing, 211189, China., Sun Q; State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University, Nanjing, 211166, China., Wang W; State Key Laboratory of Digital Medical Engineering, School of Biological Science & Medical Engineering, Southeast University, Nanjing, 211189, China., Li W; Department of Andrology, Center for Men's Health, Department of ART, Institute of Urology, Urologic Medical Center, Shanghai Key Laboratory of Reproductive Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China., Zhang W; State Key Laboratory of Digital Medical Engineering, School of Biological Science & Medical Engineering, Southeast University, Nanjing, 211189, China., Ye K; State Key Laboratory of Digital Medical Engineering, School of Biological Science & Medical Engineering, Southeast University, Nanjing, 211189, China., Wang H; State Key Laboratory of Digital Medical Engineering, School of Biological Science & Medical Engineering, Southeast University, Nanjing, 211189, China., Li Z; Feinberg School of Medicine, Northwestern University, Chicago, IL, 60611, USA., Guo Y; School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, 200242, China., Li Z; State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University, Nanjing, 211166, China.; Department of Andrology, Center for Men's Health, Department of ART, Institute of Urology, Urologic Medical Center, Shanghai Key Laboratory of Reproductive Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China.; The Affiliated Taizhou People's Hospital of Nanjing Medical University, Taizhou School of Clinical Medicine, Nanjing Medical University, Taizhou, 225300, China., Yao C; Department of Andrology, Center for Men's Health, Department of ART, Institute of Urology, Urologic Medical Center, Shanghai Key Laboratory of Reproductive Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China., Li P; Department of Andrology, Center for Men's Health, Department of ART, Institute of Urology, Urologic Medical Center, Shanghai Key Laboratory of Reproductive Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, China., Huang Y; State Key Laboratory of Digital Medical Engineering, School of Biological Science & Medical Engineering, Southeast University, Nanjing, 211189, China., Zhao X; State Key Laboratory of Digital Medical Engineering, School of Biological Science & Medical Engineering, Southeast University, Nanjing, 211189, China.
Source:
Advanced science (Weinheim, Baden-Wurttemberg, Germany) [Adv Sci (Weinh)] 2026 Jan; Vol. 13 (1), pp. e16013. Date of Electronic Publication: 2025 Nov 11.
Publication Type:
Journal Article
Language:
English
Journal Info:
Publisher: WILEY-VCH Country of Publication: Germany NLM ID: 101664569 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 2198-3844 (Electronic) Linking ISSN: 21983844 NLM ISO Abbreviation: Adv Sci (Weinh) Subsets: MEDLINE
Imprint Name(s):
Original Publication: Weinheim : WILEY-VCH, [2014]-
MeSH Terms:
Single-Cell Analysis*/methods , Transcriptome*/genetics , DNA Transposable Elements*/genetics , Transposases*/genetics , Gene Expression Profiling*/methods, High-Throughput Nucleotide Sequencing/methods ; Humans ; Male ; Animals ; Mice
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Grant Information:
82361138570 National Natural Science Foundation of China; 81827901 National Natural Science Foundation of China; 32371393 National Natural Science Foundation of China; 2022YFC2702701 National Key R&D Program of China
Contributed Indexing:
Keywords: alternative splicing; barcoded Tn5 transposase; full‐length transcriptome; multiplexed; single cell; spermatogenesis
Substance Nomenclature:
0 (DNA Transposable Elements)
EC 2.7.7.- (Transposases)
0 (Tn5 transposase)
Entry Date(s):
Date Created: 20251111 Date Completed: 20260105 Latest Revision: 20260107
Update Code:
20260130
PubMed Central ID:
PMC12766991
DOI:
10.1002/advs.202516013
PMID:
41218139
Database:
MEDLINE

*Further Information*

*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.
(© 2025 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)*