Single-Cell Nucleic Acid Analysis via Barcoded Fusion Complexes
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Solution Overview
Problem
Current methods for genetic analysis of single cells are limited by the need for large quantities of genetic material and lack the capacity for high-throughput, massively parallel analysis, making it difficult to understand biology at the single cell level and trace genetic information back to individual cells or subpopulations.
Innovation Solution
A method involving nucleic acid probes that allow for the isolation, amplification, and bulk sequencing of nucleic acid sequences from thousands of single cells, enabling the co-localization of genetic information to specific cells within a population by generating fused complexes through hybridization and PCR, and using barcode sequences to trace back sequence information to individual cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional methods like PCR or whole genome sequencing are used, then genetic analysis can be performed, but they require large quantities of genetic material (thousands to millions of cells) and cannot analyze single cells
Solution Approach 1:
The invention segments the genetic analysis process into multiple independent reaction wells, with each well containing a single cell and performing targeted amplification of specific genomic loci. This segmentation allows single-cell analysis by dividing the large-scale sequencing problem into many small, manageable parallel reactions, each requiring minimal genetic material.
Solution Approach 2:
The invention adds the dimension of spatial parallelization by arranging thousands of single-cell reactions across multiple plates and wells. Instead of analyzing cells sequentially or in small batches, the system distributes single cells across a high-dimensional space of reaction containers, enabling massively parallel analysis of thousands to millions of cells simultaneously.
2Productivity
If fluorescence-activated cell sorting (FACS) is used for single cell analysis, then hundreds of single cells can be analyzed, but the method is limited by physical constraints and cannot achieve massively parallel analysis of hundreds of thousands of cells
Solution Approach 1:
The invention creates physical copies of the single-cell analysis system across thousands of reaction wells and plates. Each well contains a complete set of reagents and performs an independent amplification reaction. This copying approach bypasses the physical limitations of FACS by distributing the analysis across a scalable array of simple, identical reaction units rather than relying on complex sorting hardware.
Solution Approach 2:
The reaction mix and protocol are designed to be universal and applicable to any single cell type. The same amplification and detection methodology works across diverse cell populations, eliminating the need for cell-type-specific optimization and enabling the system to analyze hundreds of thousands of cells from different sources and types using a single standardized platform.
3Measurement precision
If manual analysis of single cells is performed, then genetic recombination can be measured, but the process is time-consuming and tedious
Solution Approach 1:
The invention performs preliminary amplification of target genomic sequences in each single-cell reaction before pooling and sequencing. By pre-amplifying the DNA in thousands of parallel reactions, the system prepares sufficient material for downstream analysis without requiring time-consuming manual handling of individual cells during the critical measurement phases. The barcoding is also performed in advance, enabling later computational tracing.
Solution Approach 2:
The invention merges thousands of individually barcoded single-cell amplification reactions into a single pooled library for high-throughput sequencing. This combining approach maintains the ability to trace each sequence back to its parent cell through the barcode while eliminating the need for time-consuming sequential processing. The merged library can be sequenced in a single instrument run, dramatically reducing total analysis time.
4Loss of information
If conventional sequencing methods are used, then genetic information can be obtained, but it is difficult to trace sequence information back to individual cells or subpopulations
Solution Approach 1:
The invention introduces a molecular barcode as an intermediary element that links each single cell's genetic material to its identity. The barcode sequence is incorporated into the amplified DNA product during the single-cell reaction, serving as a permanent tag that travels with the genetic information through pooling and sequencing. This intermediary enables computational tracing of sequences back to their parent cells without requiring complex physical tracking systems.
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, massively parallel genetic characterization of single cells, allowing for the analysis of thousands to millions of cells and tracing genetic information back to individual cells or subpopulations, overcoming the limitations of existing techniques.
Implementation Method 1
a first probe comprising a sequence that is complementary to a first target nucleic acid subsequence, a second probe comprising a sequence that is complementary to a second subsequence of the first target nucleic acid
Implementation Method 2
amplifying the first and second target nucleic acid sequences independently, wherein the first target nucleic acid sequence is amplified using the first probe and the second probe
Data Source
AI summary
Methods and systems are provided for massively parallel genetic analysis of single cells in emulsion droplets or reaction containers. Genetic loci of interest are targeted in a single cell using a set of probes, and a fusion complex is formed by molecular linkage and amplification techniques. Methods are provided for high-throughput, massively parallel analysis of the fusion complex in a single cell in a population of at least 10,000 cells. Also provided are methods for tracing genetic information back to a cell using barcode sequences.