Cellular Molecule Barcoding Through In Situ cDNA Tagging
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Solution Overview
Problem
Existing methods for sequencing individual transcripts from cells are complex, require specialized equipment, or are limited in scalability and accuracy, especially when dealing with large samples.
Innovation Solution
A method involving the in situ reverse transcription of RNA into cDNA, followed by the ligation of nucleic acid tags to form unique barcodes, allowing for the separation and labeling of individual cells in separate reaction vessels, and repeated rounds of tagging and pooling to ensure each cell has a unique barcode.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If manual separation of individual cells into separate reaction vessels is used, then individual cell sequencing can be achieved, but the process becomes too complicated and requires specialized equipment
Solution Approach 1:
The patent introduces barcodes as intermediary molecules that mediate between individual cells and sequencing analysis. Instead of physically separating and analyzing each cell individually through complex equipment, barcodes are attached to cDNA from individual cells, allowing pooled sequencing where barcode sequences serve as identifiers linking reads back to their source cells, thereby simplifying the experimental workflow while maintaining individual cell resolution
Solution Approach 2:
The patent creates informational copies of cell identity through barcode sequences. Rather than physically isolating and sequencing each cell's complete transcriptome separately, the method generates barcode copies that represent each cell's identity, which can then be read alongside pooled cDNA sequences to reconstruct individual cell transcriptomes from bulk sequencing data
2Measurement 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 combines multiple cells' cDNA into a single pooled sample for bulk sequencing, rather than sequencing individual cells separately through microscopy. By attaching unique barcodes to each cell's cDNA before pooling, the method achieves both high-throughput processing of many cells simultaneously and accurate base identification through standard high-accuracy sequencing chemistry
Solution Approach 2:
The patent adds an informational dimension through barcode sequences that encode cell identity. This allows the system to handle high-dimensional data from many cells simultaneously through bulk sequencing, rather than being constrained to low-dimensional, sequential imaging of individual cells. The barcode dimension enables computational deconvolution of pooled signals back to individual cell sources
3Productivity
If all cDNA sequences are mixed together before sequencing, then NGS can be performed efficiently, but RNA expression cannot be linked back to individual cells
Solution Approach 1:
The patent uses barcodes as intermediary markers that travel with cDNA through the pooling and sequencing process. These barcode sequences are co-sequenced with the cDNA, serving as informational tags that enable computational association of sequencing reads with their source cells even though the physical samples are mixed together during bulk processing
Solution Approach 2:
The patent implements a feedback mechanism where barcode sequences from the pooled sequencing output provide information that feeds back into the data analysis pipeline. By reading and decoding barcode sequences from the bulk sequencing data, the system can computationally reassign reads to their source cells, thereby recovering individual cell information from pooled sequencing results
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 accurate transcriptome analysis.
Implementation Method 1
conversion of the RNA transcripts into complementary DNA (cDNA) using reverse transcription
Implementation Method 2
ligation of nucleic acid tags to form unique barcodes, allowing for the separation and labeling of individual cells
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.