Linked Ligation Adapters for Strand-Resolved DNA Sequencing
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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 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 resource-intensive.
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 methods for creating linked duplex molecules to improve sequencing accuracy by linking both strands of a duplex nucleic acid fragment.
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 resource consumption increase
Solution Approach 1:
The invention segments the sequencing process by creating multiple independent sequencing reads from a single template molecule through iterative tagmentation and amplification. Each read is an independent sampling event that can be compared to detect errors, eliminating the need to sequence multiple copies of the same template simultaneously. This segmentation approach maintains error detection capability while reducing overall sequencing resource consumption.
Solution Approach 2:
The invention performs preliminary amplification and library preparation steps before sequencing, creating multiple copies of the template with unique identifiers (barcodes) during the tagmentation process. This preliminary action ensures that sufficient material is available for multiple sequencing reads without consuming excessive sequencing bandwidth during the actual sequencing phase.
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 library preparation steps into a single integrated process. The tagmentation step simultaneously performs target enrichment and adapter ligation, eliminating the need for separate hybridization, washing, and amplification steps required by traditional target capture methods. This merging reduces workflow complexity while maintaining specificity through the use of sequence-specific guide RNAs that direct the tagmentation activity to target regions.
Solution Approach 2:
The invention uses guide RNAs as intermediaries to mediate between the target DNA and the tagmentation machinery. These guide RNAs provide sequence-specific recognition of target regions and recruit the tagmentation complex to the correct locations, achieving high specificity without requiring multiple binding and washing steps. The guide RNA acts as a molecular bridge that simplifies the overall process while maintaining precision.
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 ligation yields, simplifies workflows, reduces sequencing costs, and increases accuracy by differentiating between true variants and errors through simultaneous analysis of both strands, thereby improving base calling and alignment efficiency.
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.