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 in base calling and alignment, which are exacerbated by amplification and sequencing errors, leading to sequence misalignment and misidentification of mutations, and existing methods to mitigate these errors are costly and inefficient.
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 to facilitate targeted ligation of adapters onto double-stranded DNA, allowing for increased ligation yields and simplified workflows, and the creation of linked duplex molecules to improve sequencing accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
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 capture and ligation steps, reducing the number of amplification reactions needed while maintaining error detection capability through duplex sequencing of both DNA strands.
Solution Approach 2:
The linked ligation adapter serves multiple functions: it acts as both a capture probe for target selection and as a sequencing adapter simultaneously. This multi-functional design eliminates the need for separate capture and adapter molecules, reducing the number of copies required while achieving both target enrichment and error correction through duplex sequencing.
2Ease of operation
If target nucleic acids are captured and sequenced using current methods with single fragment seeding, then sequencing workflow is simplified, but error propagation occurs throughout the cluster
Solution Approach 1:
The patent combines both strands of the DNA duplex into a single sequencing unit with linked ligation adapters attached to both strands. This merging ensures that both strands are amplified and sequenced together, allowing error detection through comparison while maintaining a simplified single-cluster workflow without the complexity of separate amplification reactions for each strand.
Solution Approach 2:
The patent creates complementary copies of both DNA strands with linked adapters, forming a duplex sequencing unit. By sequencing both the original and complementary strands together, errors can be identified through mismatches between the two sequences, while the workflow remains simplified as a single integrated process.
3Measurement precision
If multiple binding and extension steps are involved in target capture methods, then specificity is improved, but process complexity increases
Solution Approach 1:
The patent merges target capture and adapter ligation into a single simultaneous step using linked ligation adapters. The capture probe and adapter are pre-connected, so when the probe binds to the target, the adapter is automatically positioned for ligation. This merging maintains high specificity through the capture probe while eliminating the need for separate binding and extension steps, reducing process complexity.
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 reduces sequencing errors by ensuring that both strands of a duplex DNA molecule are sequenced together, enabling higher accuracy in base calling and alignment, while also reducing costs and simplifying workflows by eliminating the need for multiple sequencing copies and additional steps.
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.