Linked Duplex Target Capture for Sequencing Error Detection
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Solution Overview
Problem
Current high-throughput genomic sequencing platforms suffer from inaccuracies due to amplification and sequencing errors, leading to sequence misalignment and misidentification of mutations, which are costly to correct and consume sequencing bandwidth.
Innovation Solution
The method involves linking two or more nucleic acid fragments, such as the sense and antisense strands of a duplex DNA molecule, to increase information density and reduce error rates by differentiating between true variants and errors through dedicated sense-specific barcodes and sequencing reads.
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 passes: a first pass sequences one strand of the duplex, and a second pass sequences the other strand. This segmentation allows error detection without requiring multiple copies or clusters, as each pass independently sequences the complementary strand, reducing bandwidth consumption while maintaining reliability
Solution Approach 2:
Instead of using multiple copies or clusters of the same template to detect errors, the invention uses complementary copying by sequencing both strands of the duplex. The second pass sequences the complementary strand, creating a copy that allows error detection through comparison without consuming additional sequencing bandwidth
2Device complexity
If only one fragment seeds a cluster in traditional sequencing, then sequencing process is simplified, but error rates increase due to propagation of sequencing or amplification errors
Solution Approach 1:
The invention merges the sequencing of both strands into a single cluster seeding process. Both strands of the duplex are sequenced in the same cluster, combining their information to detect errors. This merging approach maintains process simplicity while improving reliability through cross-validation of both strands
Solution Approach 2:
The invention provides beforehand cushioning by sequencing both strands before final base calling. The complementary strand sequencing acts as a protective measure that detects errors in the first strand before they are propagated, cushioning against the harmful effects of sequencing and amplification errors
3Reliability
If base calling and alignment algorithms are used to correct errors, then sequencing accuracy is improved, but computational resources and time increase
Solution Approach 1:
The invention performs preliminary action by sequencing both strands during the sequencing process itself, rather than relying on post-sequencing computational correction. The complementary strand sequencing is completed before base calling and alignment, providing error detection information that reduces the need for extensive computational analysis 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 by identifying errors directly from sequencing data, reduces sequencing costs, and improves sequencing throughput without the need for extensive data analysis.
Implementation Method 1
The bound universal probe is then extended using strand displacing polymerase to produce copies of the target strands
Implementation Method 2
the reactions occur under conditions that require the target specific probe to bind in order to permit binding of the universal probe
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.