Sequencing Bias Correction via Control Fragments
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Solution Overview
Problem
Current genome-wide sequencing methods, such as genome-wide bisulfite sequencing, face challenges in clinical diagnostics due to the need for large sample sizes and low sensitivity and specificity in detecting epigenetic modifications and genetic mutations, especially in heterogeneous clinical samples.
Innovation Solution
The method involves sequencing a target region and a set of polynucleotide fragments with nucleotide tags to distinguish between different nucleotides at a specific position, allowing for deep sequencing and determination of sequencing bias to improve detection sensitivity and specificity using equimolar amounts of fragments with different nucleotides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If genome-wide bisulfite sequencing is performed to detect epigenetic modifications, then detection sensitivity and specificity are improved, but sample size requirements increase and the method becomes less suitable for clinical diagnostics
Solution Approach 1:
The method segments the sequencing process by separating target region sequencing from control fragment sequencing. Control polynucleotide fragments with known sequences and defined nucleotide compositions are sequenced alongside target regions, allowing bias correction through comparison. This segmentation enables accurate detection in small samples by using controls to calibrate sequencing behavior.
Solution Approach 2:
The invention changes the parameter of nucleotide composition in control fragments to match expected target region compositions (e.g., equimolar amounts of different nucleotides). By adjusting control fragment parameters to reflect target characteristics, the method enables accurate bias measurement and correction for small clinical samples, improving detection sensitivity without requiring large sample sizes.
2Measurement precision
If deep sequencing is performed to detect nucleotides in heterogeneous polynucleotide samples, then detection accuracy is improved, but sequencing bias increases
Solution Approach 1:
The method implements feedback by using control polynucleotide fragments with known nucleotide compositions to measure sequencing bias, then applying this measured bias information to correct target region sequencing results. The control fragments provide feedback on sequencing performance, enabling accurate detection in heterogeneous samples by compensating for position-specific and nucleotide-specific biases.
Solution Approach 2:
Control polynucleotide fragments serve as intermediaries between the sequencing process and target region analysis. These fragments with defined sequences and compositions mediate the measurement of sequencing bias, allowing the system to characterize and correct bias without directly analyzing the heterogeneous target samples. The intermediaries enable indirect measurement and correction of sequencing artifacts.
3Adaptability or versatility
If genome-wide sequencing is adapted for clinical diagnostics, then diagnostic capability is improved, but the method complexity and cost increase
Solution Approach 1:
The control polynucleotide fragments serve multiple functions: they measure sequencing bias, calibrate detection sensitivity, and provide reference standards for quality control. This multi-functionality reduces the need for separate validation experiments and simplifies the overall diagnostic workflow, making genome-wide sequencing more adaptable for clinical use without proportionally increasing complexity.
Solution Approach 2:
The method performs preliminary sequencing of control fragments with known compositions before or alongside target region sequencing to establish baseline bias parameters. This preliminary action enables pre-calibration of the sequencing system, allowing accurate diagnostic detection in clinical samples without requiring complex post-hoc corrections or additional validation steps.
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 enables accurate detection of nucleotides in small sample sizes, reducing sequencing bias and enhancing the sensitivity and specificity of epigenetic and genetic mutation detection, making genome-wide sequencing more suitable for clinical diagnostics.
Implementation Method 1
A reaction mixture for sequencing the target region and the set of polynucleotide fragments may include one or more primers that hybridize specifically to the target region and one or more primers that hybridize specifically to the set of polynucleotide fragments
Data Source
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AI summary
Disclosed are methods for determining one or more nucleotides at one or more nucleotide positions of a polynucleotide sample, the polynucleotide sample comprising heterogeneous polynucleotides having different nucleotides at the nucleotide positions. The disclosed methods may be utilized to control for sequencing bias during sequencing of the polynucleotide sample. Suitable samples may include patient samples for use in diagnosing, prognosing, and treating the patient.