Single-Cell Nucleic Acid Barcoding for Low-Copy Quantification
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Solution Overview
Problem
Existing methods struggle to provide genome-wide, digital quantification of nucleic acid molecules with high dynamic range and single molecule sensitivity, especially for low copy number molecules, complicating RNA expression profiling in single cells.
Innovation Solution
A method involving tagging each nucleic acid molecule with a unique barcode sequence, amplifying and sequencing these molecules to count their presence accurately, using techniques like next-generation sequencing to determine the number of unique barcode sequences, thereby quantifying nucleic acids with high sensitivity and reducing amplification bias.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional nucleic acid detection methods are used, then detection of low copy number molecules is difficult, but the methods cannot provide accurate genome-wide quantification with high dynamic range and single molecule sensitivity
Solution Approach 1:
The patent divides the nucleic acid molecules into individually barcoded units, where each molecule receives a unique barcode sequence. This segmentation allows individual molecules to be tracked and counted separately, enabling single-molecule sensitivity and accurate quantification across the entire genome, thereby resolving the contradiction between measurement precision and detection difficulty.
Solution Approach 2:
The patent introduces barcode sequences as intermediary elements that link individual nucleic acid molecules to detectable signals. These barcodes serve as mediators between the target molecules and the detection system, allowing accurate counting and quantification of low copy number molecules without requiring direct detection of the molecules themselves, thus improving both precision and detectability.
2Measurement precision
If unique barcode sequencing is used to count nucleic acid molecules, then single molecule sensitivity is achieved, but the process complexity increases
Solution Approach 1:
The patent combines multiple functions into the barcode sequencing process: identification, counting, and quantification are merged into a single workflow. By integrating these functions, the patent achieves single-molecule sensitivity while managing process complexity through consolidation rather than multiplication of separate steps.
Solution Approach 2:
The patent uses barcode sequences as copies or representations of the original nucleic acid molecules. Instead of directly analyzing the complex molecular structures, the method creates simplified barcode copies that can be easily sequenced and counted, reducing the complexity of the detection process while maintaining single-molecule sensitivity.
3Measurement precision
If amplification is used to increase signal for low copy number molecules, then detection sensitivity improves, but amplification bias is introduced
Solution Approach 1:
The patent performs barcoding before amplification, assigning unique identifiers to individual molecules in the original sample. This preliminary action allows subsequent amplification to proceed without introducing bias, because the barcodes are already in place to track and count molecules accurately, thereby maintaining reliability while improving sensitivity.
Solution Approach 2:
The patent incorporates feedback mechanisms through barcode sequencing that provides real-time information about molecule identity and abundance. This feedback allows for accurate quantification without relying on amplification, as the barcode sequences directly report the presence and number of original molecules, eliminating amplification bias while maintaining detection sensitivity.
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
This approach enables accurate counting of nucleic acid molecules, including low copy numbers, with reduced bias, providing a comprehensive expression profile of nucleic acids in single cells.
Implementation Method 1
A nucleic acid molecule in the sample is tagged or labeled with its own unique barcode sequence
Implementation Method 2
The tagged nucleic acid molecules with their own unique barcode sequences are then amplified in the case of DNA, such as cDNA
Implementation Method 3
reverse transcribed into corresponding cDNA. Each cDNA is then amplified to produce amplicons of the cDNA
Implementation Method 4
The amplicons are then sequenced whether produced from DNA or RNA and the barcodes are identified
Data Source
AI summary
Methods and compositions for digital profiling of nucleic acid sequences present in a sample are provided.