Quantitative cfDNA Analysis via Targeted Library Preparation
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Solution Overview
Problem
Existing molecular diagnostics lack efficient methods for analyzing cell-free DNA (cfDNA) due to challenges in cloning and amplifying individual DNA molecules, targeting specific genomic loci, and discriminating true positive signals from false positives, particularly in the absence of direct tumor tissue access.
Innovation Solution
A method involving end-repair of cfDNA, ligation of adaptors, amplification of cfDNA libraries, and quantitative genetic analysis of target loci using hybridization and DNA sequencing to detect genetic variants and lesions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If traditional molecular diagnostics methods (antibody-based tests, in-situ hybridization, PCR-based tests) are used, then specific nucleotide sequences can be queried, but direct access to tumor tissues is required which is often difficult or impossible to obtain
Solution Approach 1:
The patent uses circulating tumor DNA (ctDNA) as an intermediary substance that can be accessed from blood samples instead of requiring direct access to tumor tissue. The ctDNA serves as a mediator that carries genetic information from the tumor to the bloodstream, allowing molecular analysis without tissue biopsy. This resolves the contradiction by providing a reliable molecular analysis method that does not require difficult or impossible tissue access.
2Measurement precision
If DNA sequencing is used to analyze genetic variants, then comprehensive genetic information can be obtained, but the sensitivity is insufficient to discriminate true positive test results from false positive signals
Solution Approach 1:
The patent segments the DNA analysis process into multiple independent steps: cfDNA extraction, library preparation with unique molecular identifiers (UMIs), target enrichment, sequencing, and computational analysis. Each step is optimized to minimize errors and enable discrimination of true positives from false positives. The UMI segmentation allows tracking of individual DNA molecules through the workflow, improving measurement precision and reliability.
Solution Approach 2:
The patent implements feedback mechanisms through quality control metrics and computational algorithms that compare observed variant frequencies against expected error rates. The system uses duplicate read analysis and UMI consensus building to provide feedback on signal authenticity, allowing discrimination of true positive variants from false positive sequencing errors.
3Measurement precision
If targeted sequence capture is used to enrich specific genomic loci, then sequencing depth can be increased, but the complexity of library preparation and target selection increases
Solution Approach 1:
The patent employs universal adapter sequences and standardized library preparation protocols that can be applied to any cfDNA sample regardless of the specific genetic condition being investigated. The target capture design uses universal probe structures that can be reconfigured to target different genomic regions, reducing the complexity of library preparation while maintaining high sequencing depth for targeted loci.
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 reliable and sensitive detection of genetic lesions and variants in cfDNA, overcoming the limitations of direct tissue access and providing comprehensive genetic analysis from bodily fluids.
Implementation Method 1
treating cfDNA with one or more end-repair enzymes to generate end-repaired cfDNA
Implementation Method 2
ligating one or more adaptors to each end of the end-repaired cfDNA to generate a cfDNA library
Implementation Method 3
amplifying the cfDNA library to generate cfDNA library clones
Implementation Method 4
performing a quantitative genetic analysis of one or more target genetic loci in the cfDNA library clones
Data Source
AI summary
The invention provides a method for genetic analysis in individuals that reveals both the genetic sequences and chromosomal copy number of targeted and specific genomic loci in a single assay. The present invention further provides methods for the sensitive and specific detection of target gene sequences and gene expression profiles.