Parallel Liquid-Phase Hybrid Capture for Genomic Target Regions
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Solution Overview
Problem
Current liquid-phase hybrid methods for targeted enrichment sequencing can only capture one strand of DNA molecules, leading to inefficiencies in detecting low initial amounts or low-frequency mutations, particularly in samples like liquid biopsies and small FFPE samples.
Innovation Solution
A method involving the simultaneous capture of sense and antisense strands using probe sets that overlap, allowing for bidirectional amplification and labeling, enhancing capture efficiency and sequencing depth.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If only a single strand (sense or antisense) is captured during hybridization using conventional liquid-phase hybrid methods, then the capture process is simple and cost-effective, but the capture efficiency is limited to approximately 50% of target molecules
Solution Approach 1:
The probe set is segmented into two distinct subsets: sense strand probes and antisense strand probes. Each subset targets a specific strand of the target DNA, allowing simultaneous capture of both strands through parallel hybridization reactions. This segmentation enables the system to overcome the 50% capture efficiency limitation by dividing the capture function across multiple specialized probe components.
Solution Approach 2:
The probe set achieves multi-functionality by incorporating both sense and antisense capture capabilities within a single hybridization reaction system. The same hybridization buffer and magnetic bead system can simultaneously perform both sense strand capture and antisense strand capture, eliminating the need for separate capture processes and reagent systems.
2Measurement precision
If conventional liquid-phase hybrid methods are used for targeted enrichment, then the method is cost-effective for standard samples, but it cannot effectively detect low initial amounts or low frequency mutations in challenging samples such as liquid biopsies and small FFPE samples
Solution Approach 1:
The method performs preliminary capture of target DNA strands before amplification by capturing both sense and antisense strands simultaneously in the hybridization reaction. This preliminary capture of both strands ensures that even low-abundance target molecules are efficiently recovered and made available for subsequent amplification, thereby enhancing detection sensitivity for low initial sample amounts.
Solution Approach 2:
The invention changes the key parameter of strand-specific capture by introducing antisense strand capture in addition to sense strand capture. This parameter change from single-strand to dual-strand capture fundamentally alters the capture efficiency and detection sensitivity, enabling effective detection of low-frequency mutations in challenging sample types.
3Productivity
If only sense or antisense strand is captured and then complemented by Post-PCR, then the workflow is simple, but the sequencing depth and detection capability for low frequency mutations are severely limited
Solution Approach 1:
The capture and complementation steps are merged into a single simultaneous hybridization reaction. Both sense and antisense probes hybridize to their complementary strands in the same reaction mixture, and both strands are captured together on magnetic beads. This merging eliminates the need for separate capture and complementation steps, maintaining workflow simplicity while achieving doubled capture efficiency.
Solution Approach 2:
The simultaneous capture of both sense and antisense strands maintains continuous useful action throughout the hybridization process. Both strands are captured in parallel without interruption or sequential processing, maximizing the utilization of target molecules and enabling continuous amplification and sequencing of both strands, thereby increasing sequencing depth.
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 improves the capture efficiency and sequencing depth, enabling more effective detection of low-frequency mutations and reducing costs, particularly in challenging sample types.
Implementation Method 1
capturing the target DNA using a sense strand probe set and an antisense strand probe set targeting the target DNA
Data Source
AI summary
The present invention discloses a parallel liquid-phase hybrid capture method of simultaneously capturing sense and antisense double strands of genomic target region. The parallel liquid-phase hybrid capture method disclosed by the present invention comprises: capturing the target DNA using a sense strand probe set and an antisense strand probe set targeting the target DNA to accomplish the capture of the target DNA; the sense strand probe set consists of n sense strand probes, n is greater than or equal to 1; the antisense strand probe set consists of m antisense strand probes, in is greater than or equal to 1; both the sense strand probe set and the antisense strand probe set can cover the entire sequence of the target DNA; each probe in the sense strand probe set and each probe in the antisense strand probe set contain a recognition sequence of a transcriptase and/or a recognition sequence of a sequencing primer. The experiment proves that the present invention can significantly improve the capture efficiency and detection sensitivity of the target DNA in liquid-phase hybrid, and has wide application value in the fields of cancer mutation detection, targeted medication guidance, early screening of fetal genetic defects and infant birth defects, etc.