cfDNA Copy Number Detection Using Tagged Genomic DNA Libraries
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for detecting genetic copy number changes in biological samples, particularly in cell-free DNA, lack the sensitivity to identify rare mutations at very minor allele frequencies, making it difficult to diagnose diseases like cancer effectively.
Innovation Solution
The use of adaptors with specific design features, including amplification regions, sample tags, and anchor regions, along with unique molecule identifiers, to create genomic DNA libraries that enable high-resolution detection of copy number variations in DNA samples, particularly in cell-free DNA from blood samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional SNP genotyping methods are used, then allele frequencies can be detected at common levels (100%, 50%, 0%), but they cannot detect rare mutations at minor allele frequencies significantly less than 50%
Solution Approach 1:
The method segments the DNA analysis process into targeted capture of specific genomic regions followed by deep sequencing. By dividing the genome into manageable target regions and focusing sequencing effort there, the method achieves high detection sensitivity for rare mutations without requiring large amounts of total DNA.
Solution Approach 2:
The patent uses capture probes as intermediaries to enrich target DNA sequences from the complex cell-free DNA mixture. These probes selectively bind to and concentrate the rare mutated DNA fragments, enabling their detection despite their low abundance in the total DNA sample.
2Measurement precision
If targeted capture and deep sequencing are performed to detect rare mutations at very low minor allele frequencies, then detection sensitivity improves, but the complexity of the method increases
Solution Approach 1:
The method performs preliminary enrichment of target DNA regions using capture probes before sequencing. This pre-concentration step simplifies the subsequent sequencing analysis by ensuring that the rare mutated fragments are adequately represented, reducing the need for extremely deep sequencing and simplifying the overall workflow.
Solution Approach 2:
The patent employs universal adapter sequences and standardized capture probe designs that can be applied across different target regions and samples. This multi-functionality reduces method complexity by using the same core protocols and reagents for various genomic applications, from single-gene analysis to broader genomic studies.
3Ease of operation
If only a small amount of cell-free DNA is available from blood samples, then non-invasive diagnosis is enabled, but the ability to detect rare mutations is compromised
Solution Approach 1:
The method extracts and isolates cell-free DNA from blood plasma, separating it from cellular DNA and other contaminants. This extraction enables non-invasive sampling while concentrating the available cfDNA, maximizing the information obtainable from the limited sample material.
Solution Approach 2:
Capture probes serve as intermediaries that selectively bind to and enrich target sequences within the limited cfDNA sample. This enrichment process amplifies the signal from rare mutations without requiring additional sample material, enabling sensitive detection despite the small starting amount of DNA.
Data Source
AI summary
The present invention includes compositions and methods useful for the detection of a mutational change, SNP, translocation, inversion, deletion, change in copy number, or other genetic variation within a sample of cellular genomic DNA or cell-free DNA (cfDNA). In some embodiments, the compositions and methods of the present invention provide an extremely high level of resolution that is particularly useful in detecting copy number variations in a small fraction of the total cfDNA from a biological sample (e.g., blood).