Transposase-Assisted Tagmentation for Single-Cell Library Construction
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Solution Overview
Problem
Current high-throughput single-cell transcriptome library construction technologies, particularly 5'-end library construction technologies, suffer from low cell throughput, high empty droplet rates in micro-reaction systems, and high library construction costs, leading to inefficiencies and increased sequencing costs due to the presence of 'pseudo-single cells' (doublets or multiplets).
Innovation Solution
A method involving transposase-assisted tagmentation of RNA/DNA hybrid duplexes, combined with a novel library construction process that includes multiple rounds of ligation reactions and the use of unique molecular identifiers, to enhance cell throughput and reduce empty droplet rates, thereby improving the efficiency and cost-effectiveness of 5'-end library construction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If microfluidic droplet-based cell barcoding is used for high-throughput single-cell transcriptome library construction, then cell throughput is improved, but empty droplet rate increases and library construction cost increases
Solution Approach 1:
The invention segments the library construction process into distinct stages: (1) reverse transcription in microfluidic droplets with cell-specific barcodes, (2) pooling of droplets, (3) tagmentation with transposon sequences containing unique molecular identifiers (UMIs), and (4) secondary amplification. This segmentation allows separation of cell labeling from library construction, reducing empty droplet waste while maintaining high cell throughput.
Solution Approach 2:
The invention performs preliminary cell barcoding and cDNA synthesis in microfluidic droplets before pooling and subsequent library construction. By pre-labeling cells with unique barcodes and synthesizing cDNA in advance, the system maximizes the utility of each droplet while reducing the empty droplet rate in later stages.
2Productivity
If microfluidic droplet-based cell barcoding is used for high-throughput single-cell transcriptome library construction, then cell throughput is improved, but library construction cost increases
Solution Approach 1:
The invention merges multiple functions into the transposon sequence: it serves as both the fragmentation enzyme substrate and the source of unique molecular identifiers (UMIs) and sequencing adapters. This consolidation eliminates the need for separate adapter ligation steps and reduces reagent costs while maintaining high cell throughput.
Solution Approach 2:
The transposon sequence is designed to be universal, serving multiple purposes: (1) enabling tagmentation of pooled cDNA from thousands of cells, (2) providing unique molecular identifiers (UMIs) for molecule counting, (3) incorporating sequencing adapters, and (4) facilitating library construction. This multi-functionality reduces the number of separate reagents needed, lowering library construction costs.
3Manufacturing precision
If multiple rounds of ligation reactions are used in library construction, then manufacturing precision is improved, but device complexity increases
Solution Approach 1:
The invention replaces traditional mechanical ligation reactions with transposase-mediated tagmentation. Instead of using ligase enzymes to join DNA fragments and adapters in multiple steps, the transposase directly inserts transposon sequences containing adapters and UMIs into the cDNA fragments in a single reaction, simplifying the process while maintaining precision.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method significantly increases cell throughput and reduces empty droplet rates, resulting in more cost-effective and efficient high-throughput single-cell transcriptome sequencing by minimizing the presence of 'pseudo-single cells' and optimizing library construction processes.
Implementation Method 1
the transposase can cleave or break the double-stranded nucleic acid (e.g., the hybrid double-stranded nucleic acid containing RNA (e.g., mRNA, long non-coding RNA, eRNA) chains and cDNA chains)
Implementation Method 2
the transposase complex into a pool of nucleic acid fragments, wherein the nucleic acid fragments include cDNA fragments and the sequence of the transposon chain connected to the 5' end of the cDNA fragments
Data Source
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AI summary
The present invention relates to a method for processing cells or cell nuclei to generate a pool of nucleic acid fragments and using the generated nucleic acid fragments to produce labeled nucleic acid molecules, construct nucleic acid libraries for transcriptome sequencing, or perform high-throughput sequencing of single-cell transcriptomes. Additionally, the invention encompasses the nucleic acid libraries constructed using the described method and the kits used for implementing the disclosed methods.