Targeted Sequencing Probe Extension for Digital Nucleic Acid Quantitation
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Solution Overview
Problem
Current methods for digital nucleic acid counting, such as digital PCR and the Nanostring n-counter system, face limitations in multiplexing capacity and accuracy due to random shearing of nucleic acids before sequencing, leading to biases in gene expression and copy number variation analysis.
Innovation Solution
A method involving targeted nucleic acid sequencing that generates probe extension products by hybridizing complementary probes to specific nucleic acid sequences, followed by sequencing and alignment to a reference database, allowing for precise digital measurement of nucleic acid quantities without random shearing, thereby normalizing for gene length variations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If random shearing of nucleic acids is performed before sequencing, then high-throughput analysis is achieved, but measurement precision deteriorates due to biases in gene expression and copy number variation analysis
Solution Approach 1:
The patent extracts and removes the harmful random shearing step from the sequencing workflow. Instead of randomly fragmenting nucleic acids before sequencing, the method directly sequences specific target regions using probe-based enrichment, thereby eliminating the source of bias while maintaining high-throughput capability
Solution Approach 2:
The patent introduces probe extension products as an intermediary between the original nucleic acid and the sequencing process. These probes specifically hybridize to target regions and extend to create enriched libraries, serving as a mediator that enables targeted sequencing without random shearing, thus preserving measurement precision while achieving high throughput
2Measurement precision
If targeted nucleic acid sequencing without random shearing is performed, then measurement precision is improved by normalizing for gene length variations, but device complexity increases due to probe hybridization and extension steps
Solution Approach 1:
The patent designs probe extension products that serve multiple functions simultaneously: they hybridize to specific targets, enable enrichment of target regions, provide sequencing adapters, and normalize for gene length variations. This multi-functionality reduces the need for separate processing steps, thereby limiting the increase in device complexity while maintaining improved measurement precision
Solution Approach 2:
The patent changes the key parameter from random fragmentation to probe-guided specific enrichment. By altering the fundamental approach from non-specific shearing to targeted hybridization and extension, the method achieves better precision while the complexity increase is managed through the efficiency of probe-based enrichment rather than multiple separate steps
3Measurement precision
If digital PCR or Nanostring n-counter system is used for digital nucleic acid counting, then accuracy is improved, but multiplexing capacity is limited
Solution Approach 1:
The patent transitions from the limited dimensional approach of digital PCR (single or few targets per reaction) to a high-dimensional sequencing-based approach. By using probe extension products that can be multiplexed across thousands of targets in parallel through next-generation sequencing, the method expands the capacity dimension while maintaining digital counting accuracy
Solution Approach 2:
The patent uses probe extension products as copies that can be massively replicated and sequenced. Each probe extension product serves as a template that can be amplified and sequenced digitally, allowing thousands of different targets to be counted simultaneously with the same accuracy as digital PCR, thereby dramatically increasing multiplexing capacity while preserving measurement precision
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 high-throughput, accurate digital measurement of gene expression and copy number variation by ensuring consistent sequencing of specific nucleic acid sequences across samples, reducing biases and improving multiplexing capacity.
Implementation Method 1
hybridizing complementary probes to specific nucleic acid sequences
Data Source
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AI summary
Disclosed herein are methods, compositions and kits for quantitating one or more specific nucleic acids within a plurality of nucleic acids. In some embodiments, a sequencing library is constructed from enriched probe extension products specific for the specific nucleic acids and sequenced. In some embodiments, the resulting reads are used for removing duplicate reads. In some embodiments, counting of verified probes is used to quantitate or determine the number of specific nucleic acid molecules in the starting nucleic acid sample.