Copy Number Stable Gene Amplification for CNV Detection
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Solution Overview
Problem
Current methods for detecting copy number variations (CNVs) in genomes, particularly in cancer tissues, face challenges in accurately determining copy numbers without the need for external control samples, leading to reduced sample throughput and potential erroneous results due to abnormal copy numbers in pooled normal samples.
Innovation Solution
The method involves amplifying nucleic acid samples using primers specifically designed to target copy number stable genes, which serve as internal controls, allowing for the normalization of sequencing reads and accurate determination of copy numbers within the same sample, eliminating the requirement for external controls.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If external control samples are used for CNV detection, then measurement precision is improved, but sample throughput is reduced and device complexity increases
Solution Approach 1:
The patent combines the control function and sample analysis function into a single integrated workflow. Copy number stable genes are amplified alongside target genes in the same reaction mixture, and their sequencing reads are normalized within the same dataset, eliminating the need for separate control samples and thereby increasing sample throughput while maintaining CNV detection accuracy
Solution Approach 2:
The patent makes the sequencing library multi-functional by incorporating both target genes and copy number stable genes into the same library preparation. This universal approach allows the library to serve dual purposes: detecting CNVs in target genes and providing internal normalization controls simultaneously, thus improving productivity without sacrificing measurement precision
2Measurement precision
If pooled normal controls are used, then measurement precision is improved, but reliability deteriorates due to abnormal copy numbers in pooled samples
Solution Approach 1:
The patent implements self-service by having each sample generate its own internal control through amplification of copy number stable genes. Each sample's sequencing data includes both target gene reads and control gene reads, allowing self-normalization that eliminates reliance on external pooled controls and their associated reliability issues
Solution Approach 2:
The patent introduces copy number stable genes as intermediary elements that mediate the normalization process. These stable genes serve as internal reference points within each sample, acting as intermediaries between the target genes and the normalization calculation, thereby ensuring reliable and accurate CNV detection without external controls
3Measurement precision
If matched samples are required for control, then measurement precision is improved, but device complexity and time consumption increase
Solution Approach 1:
The patent extracts the control function from external matched samples and embeds it directly within the sequencing library itself. By amplifying copy number stable genes alongside target genes in the same reaction, the control functionality is extracted from the complex external matching process and integrated into the core workflow, thereby reducing device complexity and time consumption
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 enhances sample throughput by providing internal copy number controls within the sequencing library, reducing the need for matched samples and pooled normal controls, and ensures accurate CNV determination by normalizing sequencing reads, thereby overcoming limitations of existing methods.
Implementation Method 1
the polymerization of DNA. A microwell containing a template DNA strand to be sequenced is flooded with a single species of deoxyribonucleotide triphosphate (dNTP). If the introduced dNTP is complementary to the leading template nucleotide, it is incorporated into the growing complementary strand.
Implementation Method 2
This causes the release of a hydrogen ion that triggers an ISFET ion sensor, which indicates that a reaction has occurred.
Data Source
AI summary
Provided are methods of amplifying nucleic acids. The methods include combining a nucleic acid sample and one or more amplification primers adapted to amplify a region of one or more copy number stable genes in a reaction mixture under conditions sufficient to amplify the one or more copy number stable genes. Aspects of the present disclosure further include compositions and kits that find use in practicing embodiments of the methods.