Linked Target Capture for Duplex Sequencing Error Detection
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Solution Overview
Problem
High-throughput genomic sequencing platforms suffer from inaccuracies due to errors in base calling and alignment, which are exacerbated by amplification and sequencing errors, leading to misidentification of mutations and increased sequencing costs.
Innovation Solution
The method involves linking two or more nucleic acid fragments, such as both strands of a duplex DNA molecule, to increase information density and reduce error rates by differentiating between true variants and errors through dedicated sense and antisense sequencing reads, using linked target capture probes and primers to improve specificity and accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If standard barcode sequencing methods use tens to hundreds of copies or 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 sequencing process is divided into two independent phases: a capture phase that selectively enriches for molecules containing the target sequence, and a sequencing phase that reads the sequences. This segmentation allows error detection during capture without requiring excessive sequencing bandwidth, as the capture phase can identify and retain only the correct molecules for sequencing.
Solution Approach 2:
The patent performs target capture and error detection as a preliminary action before sequencing. By using linked target capture probes that can distinguish true variants from errors during the capture phase, the system pre-filters the sample pool to contain only high-confidence molecules, thereby reducing the sequencing bandwidth required for subsequent error-free reads.
2Measurement precision
If linked target capture probes are used to selectively enrich molecules containing target sequences, then base calling accuracy is improved, but device complexity increases
Solution Approach 1:
The patent introduces linked target capture probes as intermediary molecules that mediate between the sample DNA and the sequencing process. These probes contain both a capture region that hybridizes to the target sequence and a sequencing region that can be read, thereby improving base calling accuracy without requiring complex direct detection mechanisms.
Solution Approach 2:
The system creates multiple copies of the target sequence through amplification of the captured molecules. By generating numerous identical copies during the capture phase, the system achieves high base calling accuracy through consensus reading, while the complexity is managed by using standard amplification techniques rather than complex single-molecule detection.
3Manufacturing precision
If multiple binding and extension steps are involved in linked target capture, then specificity is improved, but processing time increases
Solution Approach 1:
The patent merges the target capture and sequence reading operations into a single integrated process. The linked target capture probes are designed to simultaneously perform capture and provide sequencing information, eliminating the need for separate capture and sequencing steps and thereby reducing processing time while maintaining high specificity.
Solution Approach 2:
The capture probes are designed to continuously perform both capture and sequencing functions throughout the process. By maintaining continuous useful action where the same molecular complex performs multiple functions in sequence, the system improves specificity without significant time penalty, as the operations overlap rather than sequentially add time.
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 base calling accuracy, reduces sequencing costs, and decreases error rates by identifying errors directly from raw sequencing data without bioinformatics analysis, while maintaining high throughput.
Implementation Method 1
Linked target capture probes include a universal probe and a target specific probe wherein the reactions occur under conditions that require the target specific probe to bind in order to permit binding of the universal probe
Implementation Method 2
The bound universal probe is then extended using strand displacing polymerase to produce copies of the target strands
Implementation Method 3
which can then be amplified using PCR with universal primers
Data Source
AI summary
The invention generally relates to sequencing library preparation methods. In certain embodiments, two or more template nucleic acids are joined together by a linking molecule, such as a PEG derivative. Identical copies of a nucleic acid fragment or both strands of a duplex fragment may be linked together. The linked nucleic acids are amplified, creating linked amplicons. Emulsion PCR with linked primers creates linked template nucleic acids for seeding sequencing clusters and errors can be readily identified by their presence on only one of the linked fragments.