Randomized Cell Gating for Unbiased Sort-Seq Fluorescence Estimation
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Solution Overview
Problem
The 'sort-seq assay' method for cell sorting and sequencing introduces systematic bias and imprecision due to variability in fluorescence values, leading to inaccurate determination of cell design distributions across tubes.
Innovation Solution
Implementing randomized sorting rules based on pseudo-random numbers to allocate cells into bins, combined with multiplexed DNA sequencing, allows for more accurate estimation of mean fluorescence values by minimizing cell-to-cell variability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a histogram approach is used to sort cells into a finite set of tubes, then the sorting process is efficient and simple, but systematic bias is introduced in determining the distribution of different cell designs
Solution Approach 1:
The patent applies dynamic sorting rules where the bin assignment for each cell is determined by a combination of its fluorescence value and a pseudo-random number. This dynamic approach replaces the static histogram binning with a probabilistic assignment mechanism that adapts to individual cell measurements, reducing systematic bias while maintaining sorting efficiency
Solution Approach 2:
The patent changes the parameter space by introducing pseudo-random numbers as an additional dimension for bin assignment. Instead of using only fluorescence values to determine bin placement, the system incorporates stochastic elements that allow cells with similar fluorescence values to be distributed across multiple bins, thereby reducing the systematic bias inherent in traditional histogram approaches
2Quantity of substance
If FACS sorts many cells with different measured fluorescence values into the same bin, then the sorting capacity is high, but it becomes impossible to map DNA sequences for cell design types to the fluorescence values measured for these cells
Solution Approach 1:
The patent segments the population of cells with similar fluorescence values across multiple bins using pseudo-random assignment. Instead of consolidating all cells within a fluorescence range into a single bin, the system divides them across multiple bins probabilistically, preserving the ability to track fluorescence value information through the combination of bin assignment and sequencing data
3Device complexity
If traditional histogram bins are used with limited number of tubes, then the sorting process is simple, but the ability to differentiate between cell designs collected into the same tube is lost
Solution Approach 1:
The patent adds another dimension to the sorting process by incorporating pseudo-random number assignment alongside fluorescence value-based binning. This dimensional expansion allows the system to maintain simple physical sorting (one dimension) while adding computational differentiation capability (second dimension), thereby improving cell design differentiation without significantly increasing physical system complexity
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
The method provides unbiased mean fluorescence values for cell design groups, improving accuracy and efficiency in characterizing cell libraries by reducing systematic bias and enhancing the precision of cell type differentiation.
Implementation Method 1
Each cell design exhibits a characteristic intensity or value of fluorescence when bound to a fluorescent marker
Data Source
AI summary
Systems and methods are provided for sorting cells with distinct cell designs for characterizing a library of the cell designs. The present disclosure uses randomized sorting rules associated with bins and pseudo-random numbers to counts the cells with measured fluorescence values in one of the bins. A mean fluorescence value for a cell design group may be determined based on a ratio of cell counts of the cells associated with the cell design group across the bins. Unlike the traditional histogram-based sorting that use a mean fluorescence value of a bin, the disclosed technology determines mean fluorescence values of cell design groups for characterizing libraries of the cell design group. Use of the mean fluorescence values with unbiased “sort-seq” and a de-multiplexed sequencing using the mean fluorescence values enables characterizing libraries of cell designs with improved accuracy over traditional use of discrete histograms.