Linked Ligation Adapters for Duplex Sequencing Accuracy
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Solution Overview
Problem
Current nucleic acid sequencing technologies suffer from inaccuracies due to high error rates, misalignment, and complex workflows, particularly in target nucleic acid isolation and base calling, which are exacerbated by amplification and sequencing errors.
Innovation Solution
The use of linked ligation adapters that selectively ligate to target sequences, allowing for increased ligation yields and simplified workflows by combining target sequence selection and capture with adapter ligation, and incorporating isothermal recombinase and single-strand binding proteins for targeted ligation, enabling methods for both single and double-stranded DNA.
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 applies preliminary action by performing targeted enrichment of the target nucleic acid sequence before sequencing. The method uses probes complementary to the target sequence to capture and concentrate the specific sequence of interest from a complex sample, creating a enriched pool that contains multiple copies of the target sequence. This preliminary enrichment step ensures sufficient material for accurate sequencing and error detection without requiring excessive sequencing bandwidth, as only the enriched target sequences are sequenced rather than entire genomes or large genomic regions.
2Productivity
If current methods attach DNA fragments to solid support and amplify to form clusters, then amplification efficiency is improved, but error propagation throughout the cluster occurs
Solution Approach 1:
The patent applies segmentation by dividing the amplification process into separate stages: first, targeted enrichment of the specific nucleic acid sequence using probe hybridization; second, amplification of only the enriched target sequences; and third, sequencing. This segmentation ensures that amplification occurs only after accurate target selection, preventing error propagation from non-target sequences. The method also segments the error detection approach by using unique molecular identifiers (UMIs) that are attached to individual molecules before amplification, allowing computational distinction between true biological variants and amplification errors.
Solution Approach 2:
The patent uses probes as intermediaries between the target nucleic acid sequence and the amplification/sequencing process. These probes hybridize specifically to the target sequence, serving as a mediator that selects and enriches the desired sequence before it undergoes amplification. The probes can be designed with unique molecular identifiers that are incorporated into the amplified products, acting as intermediaries that track individual molecules through the amplification process and enable error correction during data analysis.
3Measurement precision
If base calling and alignment algorithms are used to compensate for sequencing errors, then some error correction is achieved, but quality is negatively impacted by amplification and sequencing errors
Solution Approach 1:
The patent applies preliminary action by incorporating unique molecular identifiers (UMIs) during the target enrichment and amplification stages, before sequencing occurs. These UMIs are attached to individual nucleic acid molecules and serve as preliminary error-tracking markers. During base calling and alignment, the UMIs enable computational methods to distinguish between true biological variants and errors introduced during amplification or sequencing, significantly improving base calling accuracy and alignment quality without relying solely on post-sequencing algorithms.
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 linking both strands of a duplex nucleic acid fragment, improving alignment accuracy and reducing costs through enhanced base calling and simplified workflows, while maintaining high sequencing throughput.
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.