CNV Detection via Sequencing Coverage Normalization
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Solution Overview
Problem
Current methods for detecting copy number variations (CNV) in DNA, such as FISH and aCGH, suffer from low resolution, making it difficult to detect variations in regions smaller than 5-10 Mb and 40 kb, respectively, and DNA sequencing introduces biases and errors due to amplification and short subsequence analysis.
Innovation Solution
A method involving DNA sequencing that includes purifying, fragmenting, and labeling patient DNA samples, pooling them with controls, and using computer systems to demultiplex, align, and normalize coverage data to detect CNV by comparing actual and reference coverage values, allowing for precise identification of copy number variations without demographic or phenotypic information.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If FISH is used to detect CNV, then the method is simple and visual, but the resolution is low and cannot detect regions shorter than 5-10 Mb
Solution Approach 1:
The patent replaces the mechanical/optical FISH system with a molecular sequencing-based system. Instead of using fluorescent probes and optical microscopy to detect CNV, the invention sequences short DNA segments and uses computational analysis to identify copy number variations, achieving much higher resolution without requiring complex optical instrumentation
Solution Approach 2:
The invention changes the detection parameter from optical signal intensity (fluorescence) to sequence coverage depth. By measuring the number of sequencing reads covering each genomic position, the system can detect CNV at a resolution much finer than FISH, limited only by the sequencing depth rather than optical resolution
2Measurement precision
If aCGH is used to detect CNV, then the method provides better resolution than FISH, but still cannot detect CNV of regions smaller than about 40 kb
Solution Approach 1:
The patent replaces the aCGH system that relies on physical hybridization and optical detection with a sequencing-based approach. By directly sequencing short DNA fragments and analyzing coverage through computational methods, the system achieves resolution far beyond the 40 kb limit of aCGH while maintaining relative simplicity through automated sequencing and analysis pipelines
Solution Approach 2:
The invention segments the genome into many short overlapping sequences (reads) through sequencing. This segmentation allows for high-resolution analysis of copy number variations at the base-pair level, as each short read can be independently analyzed for coverage, enabling detection of CNV smaller than 40 kb
3Measurement precision
If DNA sequencing is used to detect CNV, then resolution can be improved, but introduces biases and sampling errors due to amplification and short subsequence analysis
Solution Approach 1:
The patent implements feedback mechanisms through iterative sequencing and analysis. By sequencing multiple short segments and using computational algorithms to analyze coverage patterns, the system can identify and correct biases and sampling errors. The feedback loop allows for normalization of coverage data and detection of true CNV signals above background noise
Solution Approach 2:
The invention uses excessive sequencing depth and analyzes multiple overlapping short subsequences to overcome the limitations of individual short reads. By sequencing more segments than the minimum required and using computational methods to aggregate the data, the system compensates for amplification biases and sampling errors, improving overall reliability
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
Enables the detection of CNV with improved resolution and accuracy, overcoming the limitations of existing techniques by providing a robust and reliable method for identifying genetic variations in the genome, particularly useful for medical diagnostics.
Implementation Method 1
Each fragment may receive a molecular label that identifies the patient from whom the DNA was received and the DNA may otherwise be modified in preparation for sequencing
Data Source
AI summary
Embodiments of the invention include systems, apparatus, and methods for detecting copy number variation (CNV) in the genomes of one or more patients. Samples of DNA may be taken from several patients, and then sections of the patients' DNA may be sequenced, e.g., through a process that may include, for each patient, one or more of: purifying, concentrating, fragmenting, labeling, filtering, and amplifying that patient's DNA. Fragments from several patients may be pooled, and the fragments in the pool may be sequenced.The sequencing data is then subjected to analysis, which includes several normalization steps. The normalized data are then examined to identify CNV, which is reported.