Sequential Pseudo-Color Barcoding for Error-Corrected RNA Detection
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Solution Overview
Problem
Existing methods for transcriptomic profiling, such as single cell RNA-seq and qPCR, are labor-intensive, costly, and prone to artifacts, while in situ sequencing and smFISH face inefficiencies and noise due to low mRNA conversion and high fluorophore density, limiting the number of barcodes and data complexity.
Innovation Solution
A method involving sequential barcoding with n rounds of hybridization, where each round includes m serial hybridizations, allows for unique barcode assignment and error correction, enabling multiplex detection of molecular targets with improved accuracy and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If in situ sequencing or smFISH is used for transcriptomic profiling, then single molecule sensitivity is achieved, but mRNA conversion efficiency is low (1-10%) and noise/bias increases
Solution Approach 1:
The patent introduces an intermediary DNA template as a mediator between mRNA and detection. The mRNA is first converted to cDNA, then amplified through multiple rounds of DNA synthesis and amplification before final detection. This intermediary DNA template approach improves conversion efficiency from 1-10% to over 90% while maintaining single molecule sensitivity, resolving the contradiction between measurement precision and reliability.
2Adaptability or versatility
If spectral mRNA barcoding with multiple fluorophores is used, then more barcodes can be generated, but fluorophore density increases and data analysis complexity increases
Solution Approach 1:
The patent uses color changes through sequential hybridization rounds instead of simultaneous multi-fluorophore detection. In each round, a subset of probes with specific fluorophores hybridizes to targets, images are captured, then fluorophores are stripped and different probes are introduced. This temporal multiplexing approach generates many unique barcodes through combinatorial patterns of color appearances across rounds, reducing spatial fluorophore density and simplifying data analysis while maintaining high barcode capacity.
Solution Approach 2:
The patent segments the barcoding process into multiple sequential rounds, with each round detecting a subset of targets. Instead of using all fluorophores simultaneously, the detection is divided into phases where different fluorophore subsets are used in different rounds. This segmentation reduces the complexity of simultaneous multi-color imaging and data analysis while enabling exponential scaling of barcode capacity through combinatorial patterns across rounds.
3Adaptability or versatility
If sequential barcoding with multiple hybridization rounds is used, then more targets can be detected, but time consumption increases
Solution Approach 1:
The patent maintains continuity of useful action by implementing overlapping hybridization rounds and efficient fluorophore stripping protocols. While one round is being imaged, preparation for the next round can begin. The fluorophore stripping uses rapid chemical treatments that remove signals within minutes, allowing quick transition to the next hybridization round. This continuous workflow minimizes idle time between rounds, enabling detection of thousands of targets through many sequential rounds without linear time increase.
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
This approach achieves high-accuracy, time-efficient imaging-based transcriptomic profiling with single molecule sensitivity, overcoming limitations of existing technologies by reducing noise and bias, and enabling the detection of thousands of genes or the whole transcriptome.
Implementation Method 1
each barcoding round comprises m serial hybridizations of probes collectively bound to the N molecular targets
Data Source
Figure 1a~1c
Figure 2a~2b
Figure 2c
AI summary
The present invention, among other things, provides technologies for detecting and/or quantifying nucleic acids in cells, tissues, organs or organisms. Pre-designed barcodes are associated specific molecular targets through sequential hybridization experiments. A pseudo¬ color based barcoding scheme is developed to overcome limitations in the previous generation of the technology such as lack of visual signals that can be associated with the probes or small internal within cell when carrying out in situ experiments.. The current method can be applied to both in vitro and in situ analysis. According to the method, each barcoding round comprises multiple serial hybridizations where a small number of colored signals (that are associated with probes) are used in each hybridization experiment within a serial hybridization round. Images from each serial hybridization experiment within the same serial hybridization round are combined to form a composite image for each barcoding round. In each barcoding round, the same set of molecular targets are analyzed. After all barcoding rounds are completed, associated of the barcode with these molecular targets is completed.