Genomic Copy Number Determination via Probability Density Functions
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Solution Overview
Problem
Current methods for determining gene copy number in biological samples, such as the comparative threshold cycle (Ct) method, assume 100% efficiency in PCR processes, which can lead to inaccuracies due to variations in reaction conditions and instrumentation, and require calibration samples, whereas existing systems and methods fail to account for deviations and variations effectively.
Innovation Solution
The use of statistical models based on probability distribution functions (PDFs) to assign copy numbers to samples, providing a confidence value and eliminating the need for calibration samples, along with an interactive graphical user interface (GUI) for synchronized display and analysis of tabular and graphical data, allowing end-users to view and analyze large datasets efficiently.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If the comparative threshold cycle (Ct) method is used to determine gene copy number, then the calculation is simplified using the formula XN,t/XN,c=2−ΔΔCt, but the accuracy deteriorates due to the assumption of 100% PCR efficiency which is rarely achieved in practice
Solution Approach 1:
The patent changes the parameter of efficiency assumption from fixed 100% to variable actual efficiency values. It introduces methods to measure and calculate actual PCR efficiency for each reaction, then uses these measured efficiency parameters in the copy number calculation formula, replacing the idealized 2−ΔΔCt assumption with a more accurate efficiency-corrected calculation.
2Measurement precision
If calibration samples are used in the comparative Ct method, then the relative concentration can be calculated, but the device complexity and procedure complexity increase due to the requirement of additional calibration steps and assumptions
Solution Approach 1:
The patent extracts and removes the calibration sample requirement from the methodology. It develops an approach that calculates copy number based solely on measured PCR efficiency values from the actual samples, eliminating the need for separate calibration samples and the associated procedural complexity while maintaining accuracy.
3Reliability
If statistical models based on probability distribution functions are used to assign copy numbers, then the accuracy and reliability improve by accounting for assay deviations and variations, but the computational complexity and analysis time increase
Solution Approach 1:
The patent performs preliminary actions by pre-calculating and storing probability distribution parameters, mean values, and standard deviations during the initial data collection phase. It prepares lookup tables and pre-computed statistical metrics that can be quickly referenced during analysis, reducing the computational burden and time required when actual copy number assignments need to be made.
Data Source
AI summary
System and methods for the determination of a copy number of a target genomic sequence; either a target gene or genomic sequence of interest, in a biological sample are described. Various methods utilize a model drawn from a probability density function (PDF) for the assignment of a copy number of a target genomic sequence in a biological sample. Additionally, the methods provide for the determination of a confidence value for a copy number assigned to a sample based on attributes of the sample data. Additionally, various embodiments of an interactive graphical user interface (GUI) may provide an end-user with ready analysis of large sets of data representing a plurality of samples. In various embodiments of an interactive GUI, an end-user may be provided with a synchronized display of tabular and graphical sample data determined by an initial analysis according to a statistical model of a PDF. Such a synchronized display may enable an end-user to readily identify sample data for a subsequent analysis based on user input.