Single-Cell Multi-Omics via Microwell Dual-Bead Barcoding
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Solution Overview
Problem
Existing technologies face challenges in scaling parallel analysis of nucleic acids from the same cell, particularly in resolving copy number alterations and integrating RNA and DNA sequencing with live-cell imaging, and in multiplexed analysis of intracellular and extracellular proteins.
Innovation Solution
A microwell array device co-encapsulates individual cells with two barcoded beads, one for mRNA capture and the other for genomic DNA, enabling simultaneous RNA and DNA sequencing and linkage to live-cell imaging, using photocleavable linkers for releasing barcodes and integrating universal adapters into genomic DNA fragments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If multiple displacement amplification (MDA) is used for whole genome amplification, then high breadth of coverage is achieved, but amplification uniformity deteriorates
Solution Approach 1:
The patent changes the amplification parameters by using in vitro transposition with Tn5 transposase instead of traditional MDA, performing amplification at constant temperature (isothermal) rather than thermal cycling, and using a different chemical mechanism (transposition vs. polymerase-based amplification) to achieve both high coverage breadth and uniformity
Solution Approach 2:
The patent replaces the mechanical/chemical amplification mechanism of MDA with a transposition-based system where Tn5 transposase inserts adapters into genomic DNA fragments, enabling simultaneous amplification and fragment generation in a single isothermal reaction
2Manufacturing precision
If in vitro transposition with barcoded Tn5 transposases is used, then coverage uniformity is improved, but scalability deteriorates
Solution Approach 1:
The patent segments the workflow into distinct functional steps: cell encapsulation with beads, lysis, transposition, adapter incorporation, and sequencing library preparation, enabling optimization of each step for scalability while maintaining uniformity through the transposition mechanism
Solution Approach 2:
The patent creates a universal platform where the same microwell array device and transposition-based method can process thousands of cells in parallel, with the Tn5 transposase system serving multiple functions: adapter insertion, barcode incorporation, and amplification initiation
3Reliability
If pre-amplification is performed before library construction, then sequencing library quality is improved, but device complexity increases
Solution Approach 1:
The patent merges the adapter insertion step with the amplification step into a single transposition reaction, where Tn5 transposase simultaneously inserts adapters into genomic DNA and initiates amplification, eliminating separate pre-amplification steps and reducing overall process complexity
4Adaptability or versatility
If conventional fluorophore-labeled antibodies are used for protein analysis, then intracellular protein detection is enabled, but throughput deteriorates
Solution Approach 1:
The patent replaces the optical detection mechanism of fluorophore-labeled antibodies with a nucleic acid-based barcode system, where DNA-barcoded antibodies or aptamers bind to proteins and the barcode sequence is read by sequencing, enabling high-throughput multiplexed protein analysis
Solution Approach 2:
The patent changes the detection parameter from optical signals (fluorescence) to sequence-based identification, allowing simultaneous detection of multiple protein types through barcode sequencing rather than requiring separate fluorescent channels
5Productivity
If DNA-barcoded antibodies are used for multiplexed protein analysis, then throughput is improved, but adaptability to intracellular proteins deteriorates
Solution Approach 1:
The patent performs preliminary cell lysis before antibody binding, releasing intracellular proteins into the cytoplasmic mixture where they become accessible to DNA-barcoded antibodies, thereby enabling both high throughput and intracellular protein detection
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 scalable, simultaneous genome-wide analysis of RNA and DNA from thousands of individual cells, linking phenotypic and genotypic data for improved understanding of cellular heterogeneity and therapeutic responses.
Implementation Method 1
The oligonucleotides are attached to the bead by a photocleavable linker
Implementation Method 2
introducing a transposase into the microwell to integrate universal adapters into the gDNA
Implementation Method 3
barcoded adapters released from the DNA-barcoding bead hybridize to the universal adapters
Data Source
Figure 1A~1D
Figure 2A~2D
Figure 2E~2G
AI summary
Single-cell multi-omics by co-encapsulating a single cell with two beads, the first an RNA barcoding bead having barcoded mRNA capture primer oligonucleotides attached on the bead surface; and the second a DNA barcoding bead having two types of oligonucleotides releasably attached to the surface: (1) barcoded adapter oligonucleotides that are complementary to oligonucleotides bound to the transposase that are eventually incorporated into gDNA fragments and (2) polyadenylated barcoded oligonucleotides containing the same barcode sequence as the adapters. In addition, integrated analysis of RNA and protein, including intracellular protein, from individual cells using similar co-encapsulation of a single cell, an RNA barcoding bead, and with/without a specific or non-specific protein binding bead in a microwell, to avoid protein fixation by first lysing the cell to liberate intracellular contents, and then capturing protein either on a solid surface or in solution with barcoded affinity reagents.