Single-Cell Library Hashing for Throughput and Doublet Detection
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Solution Overview
Problem
Conventional high-throughput screens (HTS) are limited by their inability to detect subtle changes in cell state or gene expression and are hindered by cellular heterogeneity, leading to missed mechanistic insights and off-target effects, while current single-cell transcriptome sequencing (scRNA-seq) technologies are costly and require expensive reagents.
Innovation Solution
The use of hashing oligos for nuclear hashing and normalization oligos to create indexed single-nuclei and single-cell libraries, which increase sample throughput, enhance doublet detection, and reduce technical noise through combinatorial indexing and normalization methods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional high-throughput screening methods are used, then productivity is maintained, but measurement precision and detection sensitivity are insufficient to detect subtle changes in cell state or gene expression
Solution Approach 1:
The patent segments the screening process into bulk screening for initial identification and follow-up single-cell sequencing for detailed characterization. This segmentation allows each method to operate at its optimal throughput and sensitivity levels, resolving the contradiction between detection sensitivity and productivity.
Solution Approach 2:
The patent introduces an intermediary approach where bulk screening data guides targeted single-cell analysis. This intermediary step allows the system to maintain high throughput while achieving high measurement precision for subtle changes by focusing deep sequencing resources on promising candidates identified through bulk screening.
2Measurement precision
If single-cell transcriptome sequencing is performed to detect subtle changes and cellular heterogeneity, then measurement precision is improved, but cost and device complexity increase significantly
Solution Approach 1:
The patent divides the single-cell sequencing process into distinct modular steps: hashing/labeling, compartmentalization, sequencing, and data analysis. Each module can be optimized independently, reducing overall protocol complexity while maintaining high measurement precision for detecting cellular heterogeneity.
Solution Approach 2:
The patent employs universal hashing oligos and normalization oligos that can be applied across different cell types and experimental conditions. This universality simplifies the protocol by eliminating the need for cell-type-specific procedures, thereby reducing device complexity while maintaining precise detection of cellular heterogeneity.
3Productivity
If current hashing approaches with antibodies or chemically modified DNA oligos are used, then sample throughput is increased, but cost increases due to expensive reagents
Solution Approach 1:
The patent uses simple, inexpensive oligonucleotide hashing tags instead of expensive antibodies or chemically modified oligos. These disposable hashing oligos can be synthesized at low cost and used for high-throughput sample multiplexing, thereby increasing sample throughput while dramatically reducing reagent costs.
Solution Approach 2:
The patent changes the chemical parameters of the hashing reagents by using unmodified or minimally modified oligonucleotides instead of expensive antibodies or complex chemically modified DNA. This parameter change maintains the ability to multiplex samples at high throughput while reducing reagent costs by orders of magnitude.
4Productivity
If bulk assays are used to maintain low cost and high throughput, then productivity is maintained, but measurement precision decreases due to cellular heterogeneity masking subtle changes
Solution Approach 1:
The patent segments the analysis into bulk-level screening for initial identification and single-cell-level analysis for detailed characterization of subtle changes. This segmentation allows the system to maintain high throughput at the bulk level while achieving high measurement precision for subtle changes at the single-cell level.
Solution Approach 2:
The patent applies partial single-cell sequencing to a subset of samples identified through bulk screening, rather than sequencing all samples at single-cell resolution. This partial action maintains high overall throughput while achieving the measurement precision needed to detect subtle changes in the most promising samples.
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 enables high-throughput, cost-effective separation of cell populations and reduces technical noise, providing sensitive and specific molecular phenotyping for cellular heterogeneity analysis.
Implementation Method 1
The association between the hashing oligo and the isolated nuclei or cells can be non-specific, such by absorption
Implementation Method 2
The association between the hashing oligo and the isolated nuclei or cells can be non-specific, such by absorption
Data Source
AI summary
Provided herein are methods for preparing a sequencing library that includes nucleic acids from a plurality of single cells. In one embodiment, the method includes nuclear or cellular hashing which permits increased sample throughput and increased doublet detection at high collision rates. In one embodiment, the method includes normalization hashing which aids in estimating and removing technical noise in cell to cell variation and increases sensitivity and specificity.