In Situ Combinatorial Labeling for Single-Cell RNA Sequencing
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Solution Overview
Problem
Current Next Generation Sequencing (NGS) techniques are complex and difficult to perform on individual cells, leading to mixed cDNA sequences that cannot be linked back to individual cells, while alternative methods like microscopy are limited and difficult to implement.
Innovation Solution
A method involving the in situ labeling or barcoding of nucleic acids within cells using a series of nucleic acid tags and ligases to create unique barcodes for each cell, allowing for the separation, tagging, and pooling of cells to generate unique sequences for each cDNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Next Generation Sequencing is used to identify and quantify individual transcripts, then RNA expression can be measured, but the technique is too complicated to perform on individual cells and all cDNA sequences are mixed together before sequencing
Solution Approach 1:
The patent divides the sample into individual cells using microfluidic droplets, where each droplet contains a single cell and undergoes independent reverse transcription and labeling. This segmentation allows individual cell transcriptomes to be processed separately, preventing mixing of cDNA sequences from different cells while maintaining scalability through parallel processing of many droplets.
Solution Approach 2:
The patent performs preliminary labeling of individual cells with unique barcodes during the reverse transcription step, before all cDNA sequences are mixed together for sequencing. This preliminary action embeds cell-specific identifiers in the cDNA molecules themselves, enabling later computational separation and analysis of individual cell transcriptomes from the pooled sequencing data.
2Measurement precision
If manual separation of individual cells into separate reaction vessels is used, then unique labeling of transcripts from individual cells is achieved, but specialized equipment and complex procedures are required
Solution Approach 1:
The patent replaces manual mechanical cell separation with a microfluidic droplet generation system that automatically partitions cells into individual droplets based on flow dynamics. This substitution eliminates the need for specialized manual separation equipment and complex procedures, while achieving consistent single-cell partitioning through controlled fluid flow and droplet formation physics.
3Measurement precision
If microscopy is used to identify individual fluorescent bases, then sequencing can be performed, but the technique is difficult to implement and limited to sequencing a low number of cells
Solution Approach 1:
The patent creates physical copies of each cell's transcriptome as barcoded cDNA molecules that can be amplified and pooled. Instead of directly imaging individual bases in each cell, the method synthesizes complementary DNA copies with embedded barcodes, allowing billions of copies from many cells to be processed together through standard NGS workflows, dramatically increasing throughput compared to direct microscopy sequencing.
Solution Approach 2:
The patent combines cDNA from thousands of individually labeled cells into a single pooled sample for sequencing. By merging the barcoded cDNA molecules from many cells after preliminary individual labeling, the method achieves both single-cell resolution and high throughput, overcoming the limitation of microscopy methods that can only sequence a low number of cells.
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 the sequencing of RNA expression at the level of individual cells, providing a high likelihood of unique barcodes for each cell, facilitating the analysis of transcriptomes and protein quantification.
Implementation Method 1
reverse transcribing RNAs within the first plurality of cells to form complementary DNAs within the first plurality of cells
Implementation Method 2
a second plurality of nucleic acid tags are ligated to the complementary DNAs in the second plurality of cells
Implementation Method 3
providing a first plurality of nucleic acid tags to the first plurality of cells, wherein the first plurality of nucleic acid tags are hybridized to the complementary DNAs in the first plurality of cells
Data Source
AI summary
Methods of uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and/or a tissue are provided. Kits for uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and/or a tissue are also provided. The molecules to be labeled may include, but are not limited to, RNAs and/or cDNAs.