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 sequence misalignment, and existing error detection methods are costly and inefficient.
Innovation Solution
The method involves sequencing and linking two or more fragments, such as both strands of a duplex DNA molecule, to compare bases and identify errors by analyzing agreement or disagreement between the strands, reducing the need for unique tagging and increasing data accuracy through duplex sequencing.
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 patent combines both sense and antisense strands of a DNA duplex into a single sequencing cluster by linking them together. This merging approach allows error detection through strand comparison without requiring multiple separate clusters or template copies, thereby reducing sequencing bandwidth consumption while maintaining error detection capability
Solution Approach 2:
The patent introduces a new dimension of error detection by comparing complementary strands (sense and antisense) rather than relying solely on multiple identical copies. This dimensional approach to error detection leverages the complementary nature of DNA strands to identify errors through disagreement between strands, reducing the need for numerous template copies
2Productivity
If only one fragment seeds a cluster in amplification, then amplification efficiency is improved, but error propagation increases
Solution Approach 1:
The patent merges both sense and antisense strands into a single linked complex that together seeds the cluster. This dual-strand seeding maintains amplification efficiency while enabling error detection through strand comparison, as errors in one strand can be identified by agreement or disagreement with the complementary strand
Solution Approach 2:
The patent implements a feedback mechanism where the complementary strand serves as a reference to verify the accuracy of the sequencing read. By comparing bases at the same position on each strand, the system can identify errors introduced during amplification or sequencing, providing real-time error detection without compromising amplification efficiency
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.