Linked Ligation Adapters for Duplex DNA Sequencing Accuracy
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Solution Overview
Problem
Current genomic sequencing technologies suffer from inaccuracies due to high error rates, misalignment, and complex workflows in isolating and sequencing target nucleic acids, leading to misidentification of mutations and increased costs.
Innovation Solution
The use of linked ligation adapters that combine target sequence selection and capture with adapter ligation, utilizing isothermal recombinase and single-strand binding proteins for targeted ligation, and linking both strands of a duplex DNA molecule to improve ligation yields and sequencing accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard barcode sequencing methods use tens to hundreds of copies of the same template or ten to hundreds of clusters to create a sample pool for comparison, then error detection capability is improved, but cost and sequencing bandwidth consumption increase
Solution Approach 1:
The patent combines target capture and adapter ligation into a single integrated step using linked ligation adapters. The adapter is pre-linked to a capture probe, allowing simultaneous target selection and adapter attachment. This merging eliminates the need for separate amplification steps and reduces the number of clusters required for error detection, as each captured target directly receives its adapter in one step.
Solution Approach 2:
The adapter is preliminarily linked to the capture probe before the ligation reaction. This preliminary action ensures that when the capture probe binds to the target, the adapter is already in position and ready for ligation. This pre-positioning increases ligation efficiency and reduces the number of copies needed to achieve sufficient signal for error detection.
2Productivity
If current methods attach DNA fragments to solid support and perform sequential amplification steps, then cluster formation is achieved, but workflow complexity and time consumption increase
Solution Approach 1:
The patent merges target capture and adapter ligation into a single simultaneous step. The linked ligation adapter contains both the capture probe sequence and the adapter sequence connected by a removable linker. When the capture probe binds to the target, the adapter is automatically positioned for ligation, combining two previously separate steps into one, thereby reducing workflow complexity while maintaining productivity.
Solution Approach 2:
The removable linker acts as an intermediary between the capture probe and the adapter. It allows the adapter to be delivered to the target in a controlled manner during capture, then enables clean separation of the linker after ligation. This intermediary mechanism simplifies the overall workflow by providing a straightforward way to transfer the adapter without complex multi-step procedures.
3Manufacturing precision
If adapters are selectively ligated to target sequence followed by amplification with universal primers, then target selectivity is improved, but number of steps increases
Solution Approach 1:
The patent combines target capture and adapter ligation into one integrated step using the linked ligation adapter. The adapter is pre-linked to the capture probe, so when the probe captures the target, the adapter is simultaneously delivered and ligated to the target. This merging maintains high target selectivity through the capture probe while reducing the number of discrete steps compared to sequential capture and ligation procedures.
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 enhances sequencing accuracy by reducing error rates, simplifying workflows, and decreasing costs through improved ligation efficiency and duplex data analysis, allowing for higher fidelity in base calling and alignment.
Implementation Method 1
By using isothermal recombinase and single stranded binding proteins to generate strand invasion of double stranded DNA (dsDNA) with the ligation probe (similar to Recombinase Polymerase Amplification (RPA)) methods provide targeted ligation of adapters onto dsDNA.
Data Source
AI summary
The invention generally relates to capturing, amplifying, and sequencing nucleic acids. In certain embodiments, copies of the sense and antisense strands of a duplex template nucleic acid are captured using linked capture probes and multiple binding and extension steps to improve specificity over traditional single binding target capture techniques. Methods of seeding sequencing clusters with sense and antisense strands of a target nucleic acid are also disclosed including identifying the strands using sense-specific barcodes and confirming base calls using two sense-specific sequencing reads. Linked adapters may be used to increase adapter ligation selectively or efficiency and yield.