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

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Single-cell transcriptome analysis is advancing rapidly in depth and breadth.
• A key challenge is balancing cell throughput with full-length transcript coverage.
• Existing methods often struggle with resolution, cost, or time.

Purpose of the Study:

• To introduce CBTi-seq, a novel method for high-resolution, full-length single-cell transcriptome sequencing.
• To demonstrate CBTi-seq's ability to accurately reconstruct splice variants and structural variations.
• To provide a cost-effective and time-efficient alternative to current sequencing methods.

Main Methods:

• CBTi-seq utilizes Tn5 transposase for molecular assembly of combinatorial barcodes and unique molecular identifiers (UMIs).
• End-to-end sequencing enables base-pair precision reconstruction of transcripts.
• Orthogonal barcode design enhances multiplexing flexibility, while Tn5-delivered UMIs reduce bias and improve quantification.

Main Results:

• CBTi-seq achieves superior sensitivity and resolution compared to commercial methods.
• The method significantly reduces costs and processing time (approximately 5 hours).
• Robust identification of cell-type-specific alternative splicing and isoform switching was demonstrated in gene-edited and human testicular cells.

Conclusions:

• CBTi-seq provides a powerful tool for high-resolution single-cell transcriptome analysis.
• The method facilitates the study of complex biological processes like spermatogenesis.
• CBTi-seq has potential applications in reproductive development research and diagnostics.