Copy Number Variant Detection With Duplex Molecular Tags
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Solution Overview
Problem
Existing methods for detecting and quantifying rare genetic variants in heterogeneous DNA populations, such as cell-free DNA, are limited by the inability to accurately count molecules that are converted but not sequenced, leading to variable sensitivity and reduced accuracy.
Innovation Solution
A method involving the use of library adaptors with molecular barcodes to tag both ends of DNA fragments in a single reaction, allowing for the differentiation between paired and unpaired reads, enabling the estimation of unseen molecules and improving sensitivity to greater than 99.9% specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional sequencing methods are used to detect genetic variants, then the detection process can be completed, but the sensitivity is reduced due to inability to accurately count converted but unsequenced molecules
Solution Approach 1:
The DNA molecules are segmented by tagging each end with unique molecular barcodes, allowing individual molecule tracking. This segmentation enables the system to distinguish between paired reads from the same molecule and unpaired reads, thereby accurately counting converted but unsequenced molecules and improving detection sensitivity to greater than 99.9% specificity.
Solution Approach 2:
Unique molecular barcodes serve as intermediaries between the DNA molecules and the sequencing system. These barcodes are attached to both ends of each DNA fragment and enable the system to track and count individual molecules throughout the conversion and sequencing process, resolving the inability to accurately count unsequenced molecules.
2Measurement precision
If library adaptors with molecular barcodes are used to tag both ends of DNA fragments, then sensitivity is improved to greater than 99.9% specificity, but the device complexity increases
Solution Approach 1:
The system uses molecular barcodes that are copied and attached to both ends of each DNA fragment. This copying mechanism allows the system to create identical identifiers for each molecule end, enabling accurate tracking and counting without requiring complex unique structures for each tag, thus improving sensitivity while managing system complexity.
Solution Approach 2:
The library adaptors with molecular barcodes serve multiple functions: they tag the DNA fragments, enable molecule counting, and facilitate paired-read identification. This multi-functionality reduces the need for separate systems for each task, thereby improving detection sensitivity while limiting the increase in overall device complexity.
3Loss of information
If all molecules are converted and sequenced, then complete data is obtained, but the time and resources required increase significantly
Solution Approach 1:
The system performs preliminary tagging of all DNA molecules with unique molecular barcodes before sequencing. This preliminary action allows the system to identify and count all converted molecules in advance, enabling accurate estimation of unseen molecules and reducing the need for exhaustive sequencing of every molecule, thereby maintaining data completeness while reducing sequencing time.
Solution Approach 2:
The system uses the molecular barcode information as feedback to determine which molecules have been converted and sequenced. By counting paired reads with matching barcodes and comparing against the total number of tagged molecules, the system can identify unsequenced molecules and adjust the sequencing process accordingly, ensuring complete data collection while optimizing time usage.
Data Source
AI summary
Disclosed herein in are methods and systems for determining genetic variants (e.g., copy number variation) in a polynucleotide sample. A method for determining copy number variations includes tagging double-stranded polynucleotides with duplex tags, sequencing polynucleotides from the sample and estimating total number of polynucleotides mapping to selected genetic loci. The estimate of total number of polynucleotides can involve estimating the number of double-stranded polynucleotides in the original sample for which no sequence reads are generated. This number can be generated using the number of polynucleotides for which reads for both complementary strands are detected and reads for which only one of the two complementary strands is detected.