Duplex DNA Barcode Tagging for Accurate Copy Number Variant Detection
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Solution Overview
Problem
Existing methods for detecting and quantifying genetic variants in heterogeneous DNA samples, 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 and counting of paired and unpaired reads, thereby estimating the number of unseen molecules and improving sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional sequencing methods are used to detect genetic variants in heterogeneous DNA samples, then sequencing coverage is achieved, but the ability to accurately count molecules that are converted but not sequenced is lost, leading to variable sensitivity
Solution Approach 1:
The patent applies preliminary action by tagging DNA fragments with molecular barcodes before sequencing. This allows the system to track and count molecules that were converted during library preparation but not subsequently sequenced. The barcode tags are assigned to individual DNA fragments prior to sequencing, enabling later identification and counting of these unsequenced molecules through their barcode sequences, thereby resolving the contradiction between sequencing coverage and molecule counting accuracy.
Solution Approach 2:
The patent uses molecular barcodes as an intermediary element between the DNA fragments and the sequencing process. These barcodes serve as mediators that carry identification information about the original DNA fragments, allowing the system to count and track molecules that escape sequencing. The barcodes act as an intermediary recording mechanism that preserves molecule identity information without requiring direct sequencing of all fragments, thus improving both measurement precision and reliability.
2Measurement precision
If DNA fragments are tagged at both ends with library adaptors, then paired reads can be differentiated and counted, but the complexity of the tagging process increases
Solution Approach 1:
The patent applies segmentation by dividing the DNA fragment tagging process into distinct components: library adaptors with molecular barcodes are attached to specific ends of DNA fragments. This segmentation allows the system to differentiate between paired reads (both ends tagged) and unpaired reads (only one end tagged) by analyzing the presence or absence of barcode tags at each end, thereby achieving precise read differentiation while maintaining manageable process complexity through modular adaptor design.
Solution Approach 2:
The patent applies local quality by assigning different tagging characteristics to different ends of DNA fragments. Library adaptors with molecular barcodes are selectively attached to specific ends, creating local variations in tag presence that enable differentiation between paired and unpaired reads. This localized tagging strategy allows the system to maintain simple individual adaptor designs while achieving complex read pairing identification through the spatial distribution of tags along the DNA fragment.
3Quantity of substance
If molecular barcodes are used to tag DNA fragments, then the number of unseen molecules can be estimated, but the sequencing noise increases
Solution Approach 1:
The patent applies feedback by using molecular barcodes to track and quantify the efficiency of DNA fragment conversion during library preparation. By counting the number of unique barcodes detected in sequencing data versus the total number of fragments that should have been converted, the system can calculate conversion efficiency and use this feedback to estimate the number of unseen molecules. This feedback mechanism allows for correction of sequencing noise through statistical modeling that accounts for known conversion rates, thereby improving molecule estimation accuracy while compensating for sequencing noise.
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
Enhances the detection and quantification of rare DNA with a specificity greater than 99.9% by accurately counting both strands of DNA fragments, reducing sequencing noise and improving the sensitivity of genetic variant detection.
Implementation Method 1
tagging the original DNA fragments in a single reaction using a library of a plurality of different tags
Implementation Method 2
tagging the original DNA fragments in a single reaction using a library of a plurality of different tags
Implementation Method 3
each of the tags comprises a molecular barcode... allowing for the differentiation and counting of paired and unpaired reads
Implementation Method 4
input double-stranded deoxyribonucleic acid (DNA) can be converted by a process that tags both halves of the individual double-stranded molecule
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.