Molecular Barcode Reduction via Exonuclease Cleavage
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Solution Overview
Problem
Current methods for multiplex PCR-based nucleotide sequencing face challenges in reducing redundant molecular barcodes, leading to increased random errors and inefficiencies in detecting low-frequency mutations, particularly in FFPE DNA and ctDNA, due to the inability to effectively label both strands of DNA with identical barcodes.
Innovation Solution
A method involving three cycles of multiplex primer extension reactions with molecular barcode cassettes followed by the use of single-stranded DNA specific exonucleases to cleave redundant barcode regions, allowing for the reduction of redundant barcodes and removal of non-specific amplification products, thereby enhancing the tracing of original DNA molecules and improving sequencing accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiplex PCR-based amplification is used to amplify multiple target DNA regions, then productivity increases, but redundant molecular barcodes are generated leading to reduced measurement precision
Solution Approach 1:
The patent extracts and removes redundant molecular barcodes from the amplified DNA population using single-stranded DNA specific exonucleases. This selective removal of unwanted barcode variants preserves only the original molecular barcodes, thereby maintaining measurement precision while allowing multiplex amplification to proceed
Solution Approach 2:
The patent introduces single-stranded DNA specific exonucleases as intermediary enzymes that selectively degrade redundant molecular barcodes. These enzymes act as mediators between the multiplex PCR amplification process and the final sequencing analysis, cleaning up the barcode population without affecting the target DNA sequences
2Loss of information
If molecular barcodes are added onto target molecules during PCR amplification, then the ability to trace original molecules is improved, but redundant barcodes are created from single original molecules
Solution Approach 1:
The patent converts the harmful effect of redundant barcodes into a beneficial process by using the presence of single-stranded regions in redundant barcode structures as recognition sites for exonuclease degradation. The redundancy that was previously problematic now serves to create distinguishable structures that can be selectively removed
Solution Approach 2:
The patent changes the physical state and structure of molecular barcodes during the amplification process. Original barcodes maintain their double-stranded structure throughout, while redundant barcodes develop single-stranded regions that can be selectively targeted and removed by exonucleases
3Reliability
If ligation-based methods are used to add molecular barcodes, then identical barcodes can be added to both strands, but the process requires extensive time and resources
Solution Approach 1:
The patent replaces the mechanical ligation process with a biochemical amplification and selection process. Instead of using ligase enzymes to physically join barcodes to DNA strands, the method uses PCR amplification with selective exonuclease treatment to achieve the same goal of identical barcode labeling more efficiently
Solution Approach 2:
The patent performs preliminary barcode assignment during the initial PCR amplification cycles, before the redundant barcodes are generated and before sequencing occurs. The exonuclease treatment is applied in advance to remove redundant barcodes, eliminating the need for time-consuming post-amplification processing steps
4Quantity of substance
If PCR amplification is performed to increase DNA quantity, then sensitivity for low-frequency mutation detection is improved, but random errors are amplified along with the target sequences
Solution Approach 1:
The patent extracts and removes error-containing redundant barcode variants from the amplified DNA population. By selectively degrading molecules with redundant barcodes (which contain PCR errors) while preserving original barcode molecules, the method reduces the overall error rate in the sequenced data
Solution Approach 2:
The patent implements a feedback mechanism where the barcode structure itself provides information about the molecule's history. Molecules with redundant barcodes (indicating they are PCR amplification artifacts) are identified and removed, while molecules with original barcodes (indicating they represent true target sequences) are preserved for sequencing
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 significantly reduces redundant molecular barcodes, enhances the detection of rare mutations, and improves sequencing efficiency by allowing for the grouping of molecular barcodes into sense and antisense strands, thereby reducing random errors and increasing the accuracy of variant frequency analysis.
Implementation Method 1
introducing, following the third cycle, one or a mix of single-stranded DNA specific exonucleases to cleave one or more single-stranded DNA regions on a 5' terminus and a 3' terminus of double-stranded target-specific amplification DNA fragments
Data Source
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AI summary
Methods, compositions, systems and kits to introduce a controlled-number of molecular barcodes onto DNA fragments by reducing redundant molecular barcodes formed in template-dependent primer extension reactions, and to find variant frequencies from both strands of DNA The methods, compositions, systems and kits described herein may include, or include the use of, one or more single- stranded DNA specific nucleases that cleave the single-stranded regions containing unmatched base pairs in amplification products.