Single-Cell mRNA Sequencing with Dual-Tag Transcript Assembly
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Solution Overview
Problem
Current single-cell mRNA sequencing technologies are limited by their inability to sequence full-length transcripts and have low throughput, often requiring expensive equipment and causing cell damage, with existing high-throughput methods only capturing the 3' end of mRNA due to short reads and lacking effective cell tagging.
Innovation Solution
A method utilizing a microwell chip and microfluidic droplet system for single-cell separation, employing carriers with cell and molecule tags for dual labeling, enabling full-length cDNA sequencing through reverse transcription and transposase complex fragmentation, followed by assembly of short reads based on molecule tags to restore original sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If microfluidic platform and water-in-oil technology are used for single-cell mRNA sequencing, then cell detection throughput is improved, but full-length mRNA sequencing capability is lost
Solution Approach 1:
The patent divides the full-length mRNA sequencing task into multiple short-read sequencing fragments. Each fragment is tagged with molecule tags that contain positional information, allowing computational assembly of the complete transcript sequence from multiple short reads, thus recovering full-length information that would otherwise be lost.
Solution Approach 2:
The patent introduces molecule tags as intermediary elements that bridge the gap between short-read sequencing and full-length transcript reconstruction. These tags carry positional information that serves as a map for assembling short reads into complete mRNA sequences, enabling both high throughput and full-length sequencing capability.
2Productivity
If microfluidic chip is used for single-cell separation, then cell throughput is improved, but device complexity and control equipment requirements increase
Solution Approach 1:
The patent extracts the complex control requirements from the microfluidic chip by implementing a simpler alternative system using microwell chips. This extraction removes the dependency on complex control equipment while maintaining high cell throughput capability through parallel processing in multiple microwells.
Solution Approach 2:
The patent employs disposable microwell chips as a cost-effective alternative to expensive, complex microfluidic chips. The micrawell chips can be used once and then discarded, eliminating the need for expensive control equipment and reducing overall system complexity while maintaining high throughput.
3Measurement precision
If laser microdissection or flow cytometry is used for single-cell isolation, then cell isolation capability is improved, but cell activity is damaged
Solution Approach 1:
The patent creates a gentle physical copy or replica of the single-cell isolation process using microwell chips. Instead of using harsh laser or flow cytometry methods that damage cells, the micrawell system provides a soft, compatible environment that maintains cell viability while achieving precise single-cell isolation through physical separation in microwells.
4Productivity
If 3' end capture method is used for high-throughput sequencing, then sequencing throughput is improved, but transcript information completeness is reduced
Solution Approach 1:
The patent performs preliminary fragmentation and tagging of cDNA molecules before sequencing. By pre-fragmenting the cDNA and attaching molecule tags with positional information, the system enables subsequent high-throughput short-read sequencing to be efficiently assembled into complete transcripts, thus preserving information completeness while maintaining high throughput.
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
Enables high-throughput, cost-effective, and non-damaging full-length mRNA sequencing by tagging and assembling short reads using cell and molecule tags, allowing accurate determination of cell origin and transcript information.
Implementation Method 1
The recent combination of microfluidic platform and water-in-oil technology has improved the cell detection throughput of single-cell mRNA sequencing
Implementation Method 2
A method utilizing a micrawell chip and microfluidic droplet system for single-cell separation
Implementation Method 3
employing carriers with cell and molecule tags for dual labeling, enabling full-length cDNA sequencing through reverse transcription
Implementation Method 4
followed by assembly of short reads based on molecule tags to restore original sequences
Data Source
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AI summary
Disclosed is a method for obtaining a single-cell mRNA sequence. The method of the present invention comprises: (1) capturing mRNA of a cell by using a cell tagcarrier , and performing reverse transcription to obtain cDNA having a cell tag, cDNAs from the same cell having the same cell tag, and cDNAs from different cells having different cell tags; (2) obtaining multiple cDNA fragments having molecular tags by using a transposase complex and a molecular tag carrier, the fragments from the same cDNA having the same molecular tag, and the fragments from different cDNAs having different molecular tags; (3) performing high-throughput sequencing; (4) performing sequence assembly according to the molecular tags to obtain the sequence of each mRNA; and obtaining the sequence of all mRNAs of each single cell according to the cell tags. The method provided by the present invention can be used for obtaining the sequence of all mRNAs of each of a large number of single cells by means of high throughput.