Flow Cytometry Sorting of Barcoded Beads for Single-Cell Library Noise Reduction
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Solution Overview
Problem
Current methods for single cell genomic analysis suffer from significant noise due to non-specific nucleic acid contamination, particularly from capture particles not associated with cells, which complicates library preparation and reduces the integrity of the target library.
Innovation Solution
The method involves partitioning solid supports and single cells into compartments, where oligonucleotide barcodes hybridize with nucleic acid targets in companion partitions. This allows for the pooling of solid supports, followed by the removal and enrichment of noise and companion solid supports to generate a purified pool of barcoded nucleic acid molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-generated harmful factors
If exonuclease I treatment is used to remove non-specific oligonucleotides, then noise is reduced, but the integrity and yield of the target library are compromised
Solution Approach 1:
The patent extracts and removes only the harmful noise solid supports (beads) from the reaction mixture using flow cytometry-based sorting, while leaving the target library intact. This selective extraction approach eliminates non-specific nucleic acid contamination without compromising the integrity of the target library, resolving the contradiction between noise reduction and library integrity preservation.
Solution Approach 2:
The patent uses flow cytometry as an intermediary technology to selectively identify and remove noise solid supports based on their fluorescent properties. This intermediary method enables precise separation of harmful noise beads from the target library components, achieving noise reduction without compromising target library integrity.
2Object-generated harmful factors
If AMPure bead clean-up is used to reduce background signal, then noise is reduced, but the integrity and yield of the target library are compromised
Solution Approach 1:
The patent extracts and removes only the harmful noise solid supports (beads) from the reaction mixture using flow cytometry-based sorting, while leaving the target library intact. This selective extraction approach eliminates non-specific nucleic acid contamination without compromising the integrity of the target library, resolving the contradiction between noise reduction and library integrity preservation.
Solution Approach 2:
The patent replaces the mechanical AMPure bead clean-up method with a flow cytometry-based sorting approach. This substitution enables more precise and gentle removal of noise beads without the compromising effects of traditional clean-up methods, maintaining target library integrity while reducing background signal noise.
3Device complexity
If all beads are processed together in bulk, then the process is simple, but non-specific nucleic acid from noise partitions contributes significant noise
Solution Approach 1:
The patent segments the bead population into different types (noise beads, companion beads, and target-associated beads) and processes them separately. By sorting and removing noise beads in a separate step using flow cytometry, the method eliminates non-specific nucleic acid noise while maintaining the simplicity of the overall workflow. This segmentation approach resolves the contradiction between processing simplicity and noise reduction.
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 significantly reduces noise by removing non-specific oligonucleotides, thereby improving the integrity and yield of the target library, and enhances the accuracy of gene expression analysis.
Implementation Method 1
contacting the plurality of oligonucleotide barcodes with the copies of the nucleic acid target for hybridization in the companion partition
Data Source
AI summary
Disclosed herein include systems, methods, compositions, and kits for reducing non-specific noise-causing oligonucleotides in library preparations. Capture particles present in PCR reactions that are derived from partitions that did not contain a cell (e.g., noise capture particles) provide a significant source of non-specific nucleic acid and noise in single cell multiomics library preparations. In some embodiments of the methods provided herein, said noise capture particles are removed by a variety of approaches.