Single-Cell Nucleic Acid Analysis via Integrated Barcoding
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Solution Overview
Problem
Current methods for analyzing different types of nucleic acids in single cells face challenges in making processing cross-compatible, limiting simultaneous analysis and processing of various nucleic acids within the same cells.
Innovation Solution
A method involving the generation of fragmented genomic DNA and cDNA, barcoding, and sequencing to characterize cellular features, using an engineered transposase for DNA tagmentation and reverse transcription with unique molecular identifiers, allowing for simultaneous analysis of genomic DNA accessibility and RNA expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If separate processing methods are used for genomic DNA and RNA analysis, then each nucleic acid type can be analyzed with optimized protocols, but simultaneous analysis of different nucleic acids in single cells cannot be achieved
Solution Approach 1:
The patent combines separate genomic DNA analysis (ATAC-seq) and RNA expression analysis (RNA-seq) protocols into a single integrated workflow called SHARE-seq. Both nucleic acid types are processed simultaneously from the same single cell using a unified protocol that includes joint fragmentation, dual barcoding, and simultaneous library preparation, enabling concurrent analysis without requiring separate processing lines
Solution Approach 2:
The patent employs universal reagents and enzymes that can process both genomic DNA and RNA molecules. The engineered transposase system and reverse transcription components are designed to operate on both nucleic acid types using the same reaction conditions and buffers, creating a multi-functional processing platform that handles diverse nucleic acids through a single protocol
2Productivity
If different barcoding strategies are used for genomic DNA and RNA, then each nucleic acid can be uniquely identified, but cross-compatibility and simultaneous processing are lost
Solution Approach 1:
The patent introduces an intermediary dual barcode system that bridges genomic DNA and RNA identification. Each nucleic acid molecule receives a composite barcode consisting of a cell-specific identifier and a molecule-type-specific identifier, allowing simultaneous unique identification of both DNA and RNA from the same cell while maintaining cross-compatibility in the sequencing workflow
Solution Approach 2:
The barcode structure is segmented into distinct functional modules: a cell barcode portion that identifies the source cell and a molecule type barcode portion that identifies whether the molecule is genomic DNA or RNA. This segmentation allows independent optimization of each identification function while maintaining overall system compatibility and processing efficiency
3Reliability
If separate library preparations are performed for ATAC-seq and RNA-seq, then each library can be optimized for its specific nucleic acid type, but simultaneous analysis from single cells is not feasible
Solution Approach 1:
The patent modifies reaction parameters such as enzyme concentrations, buffer compositions, and incubation conditions to create optimized conditions that support both transposase-mediated DNA fragmentation and reverse transcription of RNA simultaneously. By carefully adjusting these parameters, the protocol maintains high reliability for both nucleic acid types while operating within a unified processing framework
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 accurate and simultaneous profiling of chromatin accessibility and gene expression in single cells, improving the understanding of cellular phenotypes and regulatory circuitry across diverse tissues.
Implementation Method 1
fragmenting the cellular genomic DNA and cDNA using an insertional enzyme. In some embodiments, the insertional enzyme is a transposase
Implementation Method 2
generating cDNA copies of cellular RNA molecules
Implementation Method 3
barcoding the fragmented genomic DNA and the cDNA within each cell such that the genomic DNA and the cDNA from the same cell receive the same unique cell barcode sequence
Implementation Method 4
the barcoded cDNA is isolated by capturing the affinity tag on a solid support
Implementation Method 5
the genomic DNA forms a complex with one or more proteins, and the genomic DNA is isolated by capturing the one or more proteins on a solid support
Data Source
AI summary
Provided herein include methods and compositions for analyzing nucleic acid in individual cells. In some embodiments, the methods herein include generating, within individual cells, fragmented cellular genomic DNA and cDNA copies of cellular RNA molecules, barcoding the fragmented genomic DNA and the cDNA within each cell such that the genomic DNA and the cDNA from the same cell receive the same unique barcode sequence, isolating the barcoded genomic DNA and cDNA, and characterizing one or more features of the individual cells based, at least in part, on sequencing of the isolated barcoded genomic DNA and the cDNA.