Hydrogel-Bead Encapsulation for Single-Cell DNA/RNA Co-Sequencing
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Solution Overview
Problem
Existing methods for single-cell sequencing of DNA methylation and RNA analysis are limited in throughput and require extensive use of liquid handlers, making it difficult to scale to high numbers of cells and integrate comprehensive biological interpretation of data.
Innovation Solution
A method involving encapsulation of single cells or nuclei in hydrogel beads using a microfluidic device, combined with combinatorial indexing and novel library construction chemistries, allowing for co-sequencing of DNA methylation and RNA from the same cell at a scale of 50,000-100,000 cells, using a three 96 well plate setup.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If existing single-cell sequencing methods are used, then DNA methylation and RNA can be analyzed, but the throughput is limited to tens of cells and requires extensive use of liquid handlers
Solution Approach 1:
The method segments the single-cell sequencing process by encapsulating individual cells in separate droplets containing gel beads, allowing parallel processing of thousands of cells simultaneously without complex liquid handling. Each droplet acts as an independent reaction chamber, dividing the bulk process into numerous small-scale parallel reactions.
Solution Approach 2:
Gel beads serve as intermediaries that enable combinatorial indexing within droplets. The gel beads capture and retain barcoded adapters during multiple pooling and partitioning steps, facilitating high-throughput processing without requiring complex liquid handling operations at each step.
2Productivity
If single-cell sequencing is performed at high throughput, then more cells can be analyzed, but the library complexity increases and data interpretation becomes more difficult
Solution Approach 1:
The method performs preliminary combinatorial indexing during droplet encapsulation and initial processing steps, assigning unique barcode combinations to each cell before pooling. This preliminary barcoding simplifies subsequent high-throughput processing by maintaining sample identity throughout the workflow, reducing information loss despite increased throughput.
3Measurement precision
If DNA methylation and RNA analysis are performed separately, then each modality can be optimized, but integration of data from the same cell becomes difficult
Solution Approach 1:
The method merges DNA methylation and RNA analysis into a single integrated workflow by performing both modalities within the same droplet-encapsulated cell. Unique barcode combinations are assigned to each cell, enabling simultaneous recovery and analysis of both methylation and transcriptome data from the same single cell, facilitating direct correlation between the two modalities.
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 high-throughput single-cell sequencing of DNA methylation and RNA analysis, achieving up to 100,000 cell nuclei sequencing with reduced library complexity and improved biological interpretation capabilities.
Implementation Method 1
The encapsulated cells are lysed and the acrylamide polymerized into a hydrogel
Implementation Method 2
encapsulation of single cells with lysis buffer and acrylamide monomer in an oil emulsion using a microfluidic device droplet maker
Implementation Method 3
the acrylamide polymerized into a hydrogel
Implementation Method 4
novel library construction chemistries for DNA methylation and RNA sequencing
Data Source
AI summary
Methods, compositions and systems for co-sequencing DNA methylation and RNA from the same cell are provided. Also provided herein are gel beads which allow for the compartmentation of single cell nuclei and allow for processing of the nucleic acids therein by addition of DNA barcodes to allow for combinatorial indexing (e.g., three-layer combinatorial indexing) of the nuclei, thereby allowing the parallel processing of single cells in a high throughput manner. The method, compositions, and systems provided herein are capable of providing single cell sequencing data from tens of thousands or more cells in a single parallel experiment.