Single-Cell Nucleic Acid Barcoding With Buffer-Exchange Workflow
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Solution Overview
Problem
Current methods for single cell nucleic acid analysis face limitations in conducting reactions under non-ideal conditions due to the inability to exchange reagent mixtures and perform buffer exchanges, leading to suboptimal results and the inability to analyze both mRNA and genomic DNA from the same cell in a single step.
Innovation Solution
A method that separates workflow steps for barcoding target nucleic acids, allowing optimal reaction conditions for each step, and enables simultaneous barcoding of RNA and genomic DNA from a single cell by using distinct capture oligonucleotides for each and incorporating barcodes during template switching.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If droplet-based partitioning is used for single cell analysis, then high parallel processing capability is achieved, but the ability to add reagents and perform buffer exchange is lost
Solution Approach 1:
The workflow is segmented into distinct stages: partitioning stage (droplet formation with cells and barcoded beads), isolation stage (magnetic separation of bead-containing droplets), and processing stage (buffer exchange and reactions in wells). This segmentation allows each stage to optimize for its specific requirements - high parallelism during partitioning, then flexibility during processing.
Solution Approach 2:
Barcoded magnetic beads serve as intermediaries that bridge the partitioning and processing stages. They allow cells to be captured and isolated in droplets, then transferred to wells where flexible reagent addition can occur. The beads maintain the link between single-cell partitioning and subsequent flexible processing.
2Adaptability or versatility
If small well-based partitioning is used to allow reagent addition, then flexibility in adding reagents is achieved, but buffer exchange between steps becomes impossible
Solution Approach 1:
The workflow separates the buffer exchange function from the reaction vessel. Magnetic beads enable buffer exchange during the isolation step by allowing complete replacement of droplet buffer with well buffer, while reactions occur in the same well. This segmentation solves the limitation of unable to exchange buffers in small wells.
3Manufacturing precision
If hybridization-based isolation is used to enable subsequent barcoding under ideal conditions, then barcoding efficiency is improved, but the ability to use target-specific primers is limited
Solution Approach 1:
The method segments the capture and barcoding functions. Magnetic beads provide non-hybridization-based capture via cell membrane binding, freeing the primer design from bead surface constraints. This allows use of any target-specific primer sequence without requiring attachment to bead surfaces, enabling both efficient isolation and flexible target-specific amplification.
4Manufacturing precision
If template switching is performed while cells are partitioned in droplets, then 5' end barcoding is achieved, but reaction conditions cannot be optimized for template switching
Solution Approach 1:
The workflow segments template switching from the droplet partitioning stage. Cells are lysed and mRNA is released in droplets, then bead-containing droplets are isolated and transferred to wells. Template switching and other reactions occur in wells where buffer conditions can be fully optimized, while still achieving 5' end barcoding through the captured mRNA-template switching oligo system.
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
Enables efficient generation of barcoded nucleic acids under ideal conditions, facilitating the combined analysis of mRNA and genomic DNA from the same cell, improving sample preparation and downstream applications like sequencing.
Implementation Method 1
Hybridizing said target RNA molecules to the capture oligonucleotides for said target RNA molecules, thereby obtaining target RNA molecules attached to said solid support
Data Source
AI summary
The invention provides a method which allows the separation of different workflow steps for barcoding of target nucleic acids and therefore providing optimal reaction conditions for each workflow step, especially for template switching reactions. Moreover this method provides the opportunity to perform the reactions such as barcoding reactions of two different nucleic acid molecules from one cell such as RNA and genomic DNA molecules in a single step. The method comprises the steps: (a) Providing a plurality of cells comprising target RNA molecules and at least one solid support comprising capture oligonucleotides for said target RNA molecules and barcode oligonucleotides; (b) Partitioning said plurality of cells and said solid supports such that each cell is included into a separate partition and each partition comprises a solid support; (c) Lysing said cell, thereby obtaining a mixture of target and non, target RNA molecules; (d) Hybridizing said target RNA molecules to the capture oligonucleotides for said target RNA molecules, thereby obtaining target RNA molecules attached to said solid support (e) Disrupting the partitions and separating the non-target RNA molecules from the target RNA molecules attached to said solid support (f) Generating double stranded nucleic acids from the target RNA molecules by nucleic acid synthesis, wherein the capture oligonucleotides serve as primer and the target RNA molecules serve as templates (g) Attaching the barcode oligonucleotides to the double stranded nucleic acids from target RNA molecules, thereby generating barcoded nucleic acids from target RNA molecules; Characterized in that in step a) said plurality of cells additionally comprise target genomic DNA molecules and said at least one solid support additionally comprise capture oligonucleotides for target genomic DNA molecules and in that the capture oligonucleotides for target RNA and target genomic DNA molecules are different.