Cellular Molecule Barcoding for Single-Cell RNA Sequencing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current Next Generation Sequencing (NGS) methods struggle to identify and quantify individual transcripts from cells in large samples due to the mixing of cDNA sequences, making it difficult to link RNA expression to individual cells, and alternative techniques like microscopy are limited and difficult to implement.
Innovation Solution
A method involving the separation of individual cells into reaction vessels, followed by the addition of nucleic acid tags with unique barcodes, repeated rounds of separation, tagging, and repooling to ensure each cell has a unique barcode, allowing for sequencing of RNA expression at the single-cell level.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If NGS methods are used to identify and quantify individual transcripts, then sequencing capability is provided, but individual cell resolution is lost due to mixing of cDNA sequences
Solution Approach 1:
The method segments the sequencing process by physically separating individual cells into distinct reaction vessels (e.g., wells of a microplate) before cDNA synthesis. Each cell's transcripts are processed independently in its own vessel, preventing mixing of cDNA sequences from different cells. This segmentation enables both NGS sequencing capability and individual cell resolution to coexist.
Solution Approach 2:
The method applies preliminary action by assigning unique barcodes to individual cells before the sequencing process. Each cell is labeled with a unique molecular identifier (UMI) and barcode during initial processing steps, establishing traceability before any mixing or pooling occurs. This preliminary tagging ensures that even if samples are pooled later, individual cell origin can be reconstructed.
2Measurement precision
If manual separation of individual cells into separate reaction vessels is performed, then individual cell resolution is achieved, but device complexity and ease of operation deteriorate
Solution Approach 1:
The method employs universal, commercially available microplate formats (e.g., 96-well, 384-well plates) that can be used with standard laboratory equipment. The same basic workflow can process varying numbers of cells by simply adjusting the plate format, making the system adaptable and eliminating the need for specialized single-cell sequencing equipment.
Solution Approach 2:
The method uses disposable microplates and consumables that are inexpensive and widely available. Rather than requiring expensive, specialized equipment for cell separation and processing, the approach relies on single-use plasticware that can be discarded after one experiment, reducing both equipment complexity and contamination risks.
3Manufacturing precision
If multiple rounds of separation, tagging, and repooling are performed to ensure unique barcodes, then labeling accuracy improves, but processing time increases
Solution Approach 1:
The method uses an excessive number of unique barcodes relative to the number of cells being processed. By providing far more unique barcode combinations than needed (e.g., millions of possible barcodes for thousands of cells), the system ensures unique identification with high probability in a single round of tagging, eliminating the need for multiple sequential separation and tagging cycles.
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 unique labeling and sequencing of nucleic acids from individual cells, providing a high likelihood of unique barcodes for each cell, facilitating the analysis of transcriptomes and other molecular profiles.
Implementation Method 1
addition of nucleic acid tags with unique barcodes, repeated rounds of separation, tagging, and repooling
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.