Cellular Molecule Barcoding for Single-Cell RNA Sequencing
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Solution Overview
Problem
Existing methods for sequencing individual transcripts from cells are complex, require specialized equipment, or are limited to low cell numbers, making it difficult to link RNA expression to individual cells.
Innovation Solution
A method involving the in situ reverse transcription of nucleic acids with unique barcodes, followed by repeated separation, tagging, and repooling of cells to generate unique barcodes for each cell's cDNA, allowing for sequencing at the single-cell level.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Next Generation Sequencing is used to identify and quantify individual transcripts, then transcript identification and quantification capability is improved, but the complexity of the procedure increases and individual cell linkage is lost
Solution Approach 1:
The patent divides the sample into individual cell compartments, with each cell processed separately to maintain cell identity. This segmentation allows NGS to be applied to individual cells while preserving the ability to link transcripts back to their cell of origin through cell-specific barcodes.
Solution Approach 2:
The patent introduces cell-specific barcodes as intermediary elements that link individual cells to their transcripts. These barcodes are incorporated into cDNA during reverse transcription, serving as mediators that enable traceability of transcripts to their parent cells while allowing bulk processing.
2Measurement precision
If manual separation of individual cells into separate reaction vessels is performed, then single-cell transcript sequencing capability is improved, but the equipment requirements and operational complexity increase
Solution Approach 1:
The patent combines multiple cells into a single reaction vessel for bulk processing while maintaining cell-specific barcodes. This merging approach eliminates the need for complex manual separation equipment while preserving single-cell resolution through barcode identification.
Solution Approach 2:
The patent uses cell-specific barcodes as informational copies that represent each cell's identity. Instead of physically separating cells, the barcode information is copied into the cDNA, allowing virtual separation and identification without complex physical manipulation equipment.
3Measurement precision
If microscopy is used to identify individual fluorescent bases, then single-cell sequencing capability is improved, but the number of cells that can be sequenced is limited
Solution Approach 1:
The patent merges multiple cells into bulk reactions while maintaining individual cell identification through barcodes. This approach enables high-throughput processing of many cells simultaneously, dramatically increasing productivity compared to microscopy-based single-cell methods.
Solution Approach 2:
The patent replaces the mechanical/optical system of microscopy with a biochemical barcode system. Instead of using microscopes to visually identify individual bases in single cells, the method uses sequence-based barcodes that can be read through standard NGS, enabling parallel processing of thousands 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 identification and quantification of RNA expression at the level of individual cells, providing a high likelihood of unique barcodes for each cell, facilitating transcriptome analysis.
Implementation Method 1
conversion of the RNA transcripts into complementary DNA (cDNA) using reverse transcription
Implementation Method 2
binding the adapter sequences within each of the at least two primary aliquots with the provided primary nucleic acid tags
Data Source
AI summary
Methods of uniquely labeling or barcoding molecules within a cell, a plurality of cells, and/or a tissue are provided. Kits for uniquely labeling or barcoding molecules within 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, cDNAs, DNAs, proteins, peptides, and/or antigens.