Single-Cell Multiomic Sequencing With Unified Barcode Amplicons
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Solution Overview
Problem
Current multiomic approaches for studying human cells face challenges due to the complexity of systems and limitations in high-throughput, single-cell analysis of DNA, RNA, and protein detection and characterization, particularly in small biological samples.
Innovation Solution
A method for simultaneous targeted detection and sequencing of DNA, RNA, and protein from a single cell using a multiomic detection and characterization strategy that involves tagging proteins with antibodies, reverse transcribing RNA, releasing DNA from the cell nucleus, and incorporating a unique cell identifier (barcode) into each analyte, followed by droplet-based PCR and sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiomic approaches are used to study analytes in human cells, then understanding and therapy development are enhanced, but system complexity and technical limitations increase
Solution Approach 1:
The method segments the multiomic analysis into distinct functional modules: (1) antibody tagging for protein detection, (2) reverse transcription for RNA conversion, (3) nuclear release for DNA extraction, and (4) barcode incorporation for cell identification. Each module operates independently but contributes to the integrated multiomic profile, reducing overall system complexity while maintaining comprehensive detection capability.
Solution Approach 2:
The patent employs universal barcoding mechanisms that can identify cells across all three omic layers (DNA, RNA, protein) simultaneously. The same barcode system is used to track cellular identity throughout the entire multiomic analysis workflow, allowing a single system to handle diverse analyte types without requiring separate identification systems for each omic layer.
2Measurement precision
If single-cell analysis is performed, then cellular heterogeneity is resolved, but the amount of biological sample available for analysis is limited
Solution Approach 1:
The method incorporates barcodes into antibodies, reverse transcription primers, and nuclear release reagents before they encounter the single cells. This preliminary tagging ensures that when the single cell is lysed and its contents released, the barcodes are already present and ready to be amplified and detected, maximizing the information obtained from the limited single-cell material without requiring additional sample collection steps.
3Measurement precision
If targeted detection of specific analytes is implemented, then detection specificity is improved, but the ability to detect multiple analyte types simultaneously is reduced
Solution Approach 1:
The patent applies local quality by assigning different barcode sequences to different antibody specificities, RNA targets, and DNA regions. Each analyte type receives specialized targeting reagents with unique barcodes, allowing simultaneous detection of multiple specific analytes within the same single-cell sample. The local customization of barcode assignments maintains detection specificity while enabling broad multi-analyte coverage.
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 analysis of DNA, RNA, and protein, allowing for customized detection of particular analytes and identification of cell clusters based on sequencing results.
Implementation Method 1
performing a protease digest on the encapsulated cell drop with the protease to produce a cell lysate
Implementation Method 2
providing a reverse transcriptase and performing a reverse transcription reaction
Implementation Method 3
performing a PCR reaction to attach the cell barcodes to the DNA targeted amplicons, RNA targeted amplicons, and protein tag amplicons
Data Source
AI summary
Provided herein are methods and systems for the simultaneous targeted detection and sequencing of DNA, RNA, and Protein. In typical embodiments, the DNA, RNA, and Proteins are detected, characterized, and sequenced using just a single mammalian cell. One embodiment of detecting and characterizing DNA, RNA, or protein from a mammalian cell includes encapsulating a single cell in a drop and performing a protease digest on the encapsulated cell drop, performing a reverse transcriptase reaction; performing a droplet merger with barcoding PCR reagents and barcoding beads; performing a PCR reaction to attach the cell barcodes to the DNA targeted amplicons, RNA targeted amplicons, and protein tag amplicons, where all amplicons from the same emulsion contain the same cell barcode; and detecting and characterizing a DNA, RNA, or protein amplicon by sequencing the cell barcode incorporated into each amplicon.