Single-Cell Multibody Sequencing Through Full-Length DNA Assembly
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Solution Overview
Problem
Existing sequencing technologies face challenges in accurately and efficiently sequencing long DNA fragments for multiome analysis of single cells, particularly in understanding complex genomic structures and cellular heterogeneity, with high costs and low quality.
Innovation Solution
A method involving multi-ligation assembly of DNA fragments, followed by selective circularization and removal of non-circular DNA using exonuclease, and optionally linearization with CRISPR/Cas nucleases, to enhance full-length sequencing efficiency and accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If second-generation NGS technologies (Illumina or IonTorrent) are used for sequencing, then high-throughput and cost-effectiveness are achieved, but the read length is limited to 100-500 bp which restricts analysis of complex genomic structures
Solution Approach 1:
The patent applies segmentation by dividing long DNA molecules into multiple shorter fragments that can be sequenced by second-generation NGS technologies. These fragments are then computationally assembled into complete long-read sequences, effectively overcoming the read length limitation while maintaining high throughput capability.
Solution Approach 2:
The patent uses computational assembly algorithms as an intermediary process between short-read sequencing and long-read analysis. This intermediary step combines multiple short reads into reconstructed long sequences, enabling the analysis of complex genomic structures without requiring actual long-read sequencing technology.
2Length of moving object
If third-generation long-read sequencing methods are used, then complex genomic structures can be analyzed, but high costs and limitations in resolving cellular heterogeneity arise
Solution Approach 1:
The patent segments the complex third-generation sequencing process into simpler components: DNA fragmentation, adapter ligation, and computational assembly. This segmentation allows the use of simpler, more cost-effective second-generation sequencing technologies while achieving long-read analysis capabilities through bioinformatics.
Solution Approach 2:
The patent creates multiple copies of DNA fragments with standardized adapters ligated to both ends. These copied and standardized fragments can be amplified and sequenced using high-throughput second-generation platforms, effectively copying the functionality of third-generation sequencing at lower cost and with better scalability for cellular heterogeneity studies.
3Productivity
If conventional sequencing methods are used for multiome analysis of single cells, then sequencing can be performed, but low quality and high cost persist
Solution Approach 1:
The patent performs preliminary actions by ligating specific adapters to both ends of DNA fragments before sequencing. This preliminary adapter ligation enables subsequent selective amplification and assembly steps, improving sequencing quality by ensuring proper fragment orientation and enabling computational reconstruction of long sequences from short reads.
Solution Approach 2:
The patent changes key parameters of the sequencing workflow: instead of sequencing native long DNA molecules directly, it fragments DNA to optimal sizes for second-generation sequencing, ligates standardized adapters, and uses computational assembly to reconstruct long sequences. This parameter transformation maintains sequencing quality while improving efficiency and reducing cost.
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
Improves the efficiency and error rate of full-length sequencing, enabling comprehensive analysis of single-cell multiomes, including gene isoform detection and cancer-specific analysis, with potential applications in disease diagnosis and targeted cancer therapies.
Implementation Method 1
a step of selectively circularizing DNA molecules in the library through a multi-ligation assembly reaction of DNA fragments
Implementation Method 2
a step of removing DNA fragments other than the circular DNA using an exonuclease
Implementation Method 3
selectively linearizing circular DNA containing target sequences to be removed using a CRISPR/Cas nuclease
Data Source
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AI summary
The present invention relates to a single cell multibody full-length sequencing analysis technique. The present invention is a development study to overcome the limitations of research on low quality and cell heterogeneity of multiplex full-length sequencing of a single cell, wherein an assembly method that allows the combination of multiple DNA fragments is used to greatly improve the efficiency and error rate of full-length sequencing, and the full-length sequencing of a single cell multibody is completed and thus can be greatly used for gene isoform expression level analysis, mutation detection, or cancer cell-specific multibody analysis.