Linked Ligation for Target Capture and Adapter Attachment
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Solution Overview
Problem
Current genomic sequencing technologies suffer from inaccuracies due to high error rates in base calling and sequencing, leading to sequence misalignment and misidentification of mutations, and existing methods for isolating target nucleic acids are complex and error-prone, with strategies to mitigate errors being costly and inefficient.
Innovation Solution
The use of linked ligation adapters that combine target sequence selection and capture with adapter ligation, employing isothermal recombinase and single-strand binding proteins to facilitate targeted ligation of adapters onto double-stranded DNA, and linking both strands of a duplex nucleic acid fragment for sequencing, thereby increasing ligation yields and reducing errors.
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 sequencing cost and bandwidth consumption increase
Solution Approach 1:
The invention segments the sequencing process by creating multiple independent template copies with unique barcodes that can be processed in parallel. Each template copy contains a unique molecular identifier (UMI) that allows traceability to the original target molecule, enabling error detection through consensus building without requiring excessive sequencing depth.
Solution Approach 2:
The invention creates multiple copies of template nucleic acids during the amplification process, where each copy carries a unique barcode/UMI. These copies serve as redundant information sources that can be compared to identify and correct sequencing errors, replacing the need for traditional methods that require tens to hundreds of clusters.
2Measurement 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 invention merges the target capture and adapter ligation steps into a single integrated process. The capture probe contains both the target-specific binding region and the adapter sequence, allowing simultaneous target recognition and adapter attachment in one step, thereby reducing process complexity while maintaining high specificity.
Solution Approach 2:
The capture probe is designed with multi-functionality, serving both as a target-specific binder and as an adapter carrier. This universal design allows the same probe structure to perform multiple functions: target recognition, signal transduction, and sequencing adapter provision, simplifying the overall workflow.
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, improving alignment efficiency, and decreasing sequencing costs through simplified workflows and direct loading of linked duplex molecules onto sequencing platforms.
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.