Single-Cell Trajectory Analysis via Detergent Lysis
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Solution Overview
Problem
Current single-cell analysis methods, such as ATAC-seq, require various biological reagents like proteases and transposases, which complicate the workflow, lead to noisy sequence reads, and are costly, especially when analyzing large numbers of cells.
Innovation Solution
A two-step workflow for single-cell analysis that minimizes the use of transposases and proteases by encapsulating cells in an emulsion with reagents that cause lysis, followed by reverse transcription and nucleic acid amplification, allowing for sequencing of RNA and chromatin-accessible DNA without the need for transposases or proteases.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If transposases and proteases are used for single-cell analysis, then chromatin accessibility can be analyzed, but the workflow becomes complicated and costs increase
Solution Approach 1:
The patent extracts and removes transposases and proteases from the single-cell analysis workflow. Instead of using Tn5 transposase for chromatin accessibility analysis, the invention uses a simplified detergent-based lysis approach that directly exposes accessible chromatin regions without requiring enzymatic processing, thereby eliminating workflow complexity while maintaining analytical capability
Solution Approach 2:
The patent replaces expensive biological reagents (transposases and proteases) with inexpensive chemical reagents (detergents). This substitution dramatically reduces consumable costs and simplifies the workflow by eliminating the need for complex enzymatic reactions, making single-cell analysis more accessible and scalable
2Reliability
If transposases and proteases are used for single-cell analysis, then chromatin accessibility can be analyzed, but sequence reads become noisy
Solution Approach 1:
The patent removes transposases and proteases from the protocol, eliminating the sources of noise and artifacts they introduce. The detergent-based lysis method provides cleaner sequence reads by avoiding enzymatic variability and off-target effects, thereby improving measurement precision while maintaining chromatin accessibility analysis capability
Solution Approach 2:
The patent uses a simplified chemical lysis approach that accurately captures chromatin accessibility states without the noise introduced by biological reagents. This method produces cleaner, more reliable sequence reads that better represent the true chromatin state, improving the fidelity of the measurement
3Reliability
If various biological reagents are used for single-cell analysis, then chromatin accessibility can be analyzed, but costs significantly increase
Solution Approach 1:
The patent replaces expensive biological reagents (transposases and proteases) with inexpensive chemical detergents. This substitution dramatically reduces consumable costs per cell while maintaining the ability to perform chromatin accessibility analysis, making the technique feasible for large-scale single-cell studies
Solution Approach 2:
The patent extracts and eliminates expensive biological reagents from the protocol, retaining only the essential detergent-based lysis step. This removal of costly components significantly reduces the per-cell analysis cost while preserving the core functionality of chromatin accessibility analysis
4Reliability
If proteases are used for cell lysis, then chromatin accessibility can be analyzed, but the workflow requires more consumables
Solution Approach 1:
The patent removes proteases from the cell lysis protocol, eliminating the need for complex enzymatic digestion steps. The simplified detergent-based approach requires fewer consumables and reduces workflow complexity, making the protocol easier to perform while maintaining chromatin accessibility analysis capability
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 simplifies the single-cell analysis workflow, reduces costs, and provides accurate sequence reads by minimizing the use of costly reagents, enabling the prediction of cell trajectories and states.
Implementation Method 1
exposing the cell to reagents that cause the cell to lyme. In various embodiments, the reagents include detergents for lysing the cell
Implementation Method 2
RNA transcripts are reverse transcribed to generate corresponding cDNA
Implementation Method 3
the reaction mixture is used to perform a nucleic acid amplification reaction
Data Source
Figure 1
Figure 2
Figure 3A~3C
AI summary
Single-cell analysis using combined RNA sequencing of RNA transcripts and DNA sequencing of chromatin-accessible DNA is performed to determine trajectories of single cells. Individual cells are encapsulated and lysed using reagents that do not include proteases or transposases. Cell lysates include RNA transcripts and packaged DNA (e.g., DNA packaged as chromatin). Segments of DNA in the packaged DNA are primed, amplified, and sequenced to generate sequence reads of the chromatin-accessible DNA. RNA transcripts are reverse transcribed to generate cDNA which is then primed, amplified, and sequenced to generate sequence reads. Sequence reads from the RNA-seq and DNA-seq reveal different states of cells and therefore, are useful for predicting cell trajectories.