Synthetic Internal Standards for Low-VAF Lung Cancer NGS
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Solution Overview
Problem
Current Next Generation Sequencing (NGS) methods struggle with imprecision, particularly in measuring low-frequency mutations associated with lung cancer risk due to technical errors, stochastic sampling, and DNA damage, leading to challenges in accurately identifying lung cancer risk and guiding targeted therapy.
Innovation Solution
Development of kits and methods that include synthetic internal standards to measure low Variant Allele Frequencies (VAF < 1%) in lung cancer driver genes, using multiplex PCR and hybrid capture techniques to enhance the accuracy of NGS in normal airway epithelial cells, enabling detection of mutations as low as 0.05% VAF.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional NGS methods are used to measure mutations, then the process is simple and fast, but the measurement precision deteriorates due to technical errors and inability to accurately detect low-frequency mutations
Solution Approach 1:
The patent introduces synthetic internal standards as intermediary molecules that serve as reference controls throughout the NGS process. These internal standards with known sequences and frequencies allow for calibration and correction of technical errors, enabling accurate measurement of low-frequency mutations (VAF < 1%) by providing a baseline for comparison against which true biological variants can be distinguished from technical artifacts
Solution Approach 2:
The patent changes the parameter of measurement sensitivity by optimizing library preparation protocols, sequencing depth, and variant calling thresholds. By adjusting these parameters and incorporating internal standards at known frequencies, the system achieves reliable detection of mutations at variant allele frequencies below 1%, transforming the measurement capability from detecting only high-frequency variants to accurately quantifying low-frequency variants
2Measurement precision
If NGS methods are used to detect low-frequency mutations, then diagnostic precision improves, but the reliability deteriorates due to stochastic sampling errors and DNA damage
Solution Approach 1:
The patent implements feedback mechanisms by incorporating internal standards that provide continuous reference information throughout the experimental process. The known frequencies and sequences of internal standards allow for real-time assessment and correction of stochastic sampling errors and DNA damage effects, enabling the system to distinguish true biological variants from technical artifacts and maintain reliable results
Solution Approach 2:
The patent applies preliminary actions by spiking internal standards into samples before library preparation and sequencing. This pre-calibration approach allows for proactive correction of potential errors from stochastic sampling and DNA damage, rather than attempting to correct them after the fact, thereby maintaining both precision and reliability
3Productivity
If standard NGS protocols are used, then the process is straightforward and quick, but measurement precision deteriorates due to PCR amplification errors and sequencing errors
Solution Approach 1:
The patent uses internal standards as intermediary reference molecules that are processed alongside sample DNA through the entire NGS workflow including PCR amplification and sequencing. These internal standards with known sequences allow for identification and correction of PCR amplification errors and sequencing errors, maintaining measurement precision without sacrificing process efficiency
Solution Approach 2:
The patent creates multiple copies of internal standard molecules with known sequences and frequencies, which are then distributed throughout the sample library. These copied reference molecules serve as numerous control points that enable detection and correction of errors introduced during PCR amplification and sequencing, maintaining precision across the entire batch of samples
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
The solution allows for more accurate stratification of lung cancer risk, reducing the need for unnecessary screening and guiding targeted therapy by identifying low-frequency mutations in non-invasive specimens, thereby improving diagnostic precision and reducing costs.
Implementation Method 1
PCR primers for each target gene
Implementation Method 2
DNA capture probes for each target gene
Data Source
AI summary
Kits and methods for diagnosing risk of developing lung cancers and uses thereof are described.