Duplex DNA Barcoding for Rare Mutation Sequencing Accuracy
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Solution Overview
Problem
Conventional next-generation sequencing (NGS) approaches struggle to accurately detect rare mutations due to high error rates and inefficient duplex recovery, particularly in limited DNA samples like cell-free plasma DNA, limiting their clinical applicability.
Innovation Solution
A method for generating duplex sequencing libraries with molecular barcodes on both strands of DNA, followed by strand-specific PCR enrichment without hybridization capture, enabling accurate detection of mutations by requiring alterations on both strands.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional NGS approaches are used, then sequencing can be performed, but the error rate is too high to allow confident detection of rare mutations
Solution Approach 1:
The invention segments the DNA into individual molecules and tags each with a unique molecular identifier (UMI). This allows tracking of each original molecule's progeny separately, enabling distinction between true mutations and sequencing errors through consensus building within families of reads derived from the same original molecule.
Solution Approach 2:
The invention creates multiple redundant copies (progeny) of each original DNA molecule through PCR amplification, with each copy carrying the same UMI. By sequencing these copies and requiring consensus among them to call a mutation, the method effectively filters out random sequencing errors while preserving true mutations.
2Measurement precision
If molecular barcoding is used to detect rare mutations, then sequencing errors can be recognized, but it is challenging to convert a large fraction of initial template molecules to duplex molecules with the same barcode on each strand
Solution Approach 1:
The invention performs preliminary tagging of DNA ends with adaptors containing UMIs before fragmentation and amplification. This preliminary action ensures that both strands of each original duplex inherit the same UMI information, enabling efficient duplex reconstruction without requiring complex post-amplification processing.
Solution Approach 2:
The invention uses forked adapters as intermediaries that facilitate the attachment of UMIs to both strands of the DNA duplex. These adapters serve as mediators that carry the UMI information through the amplification process, enabling efficient conversion of template molecules into duplex molecules with matched barcodes.
3Quantity of substance
If hybridization capture is used for target enrichment, then large regions can be enriched, but it does not scale well for small target regions and exhibits poor duplex recovery
Solution Approach 1:
The invention extracts and amplifies specific target regions using PCR with primers that bind to the adaptors and target-specific sequences. This extraction approach via PCR amplification is more efficient than hybridization capture for small target regions, as it directly amplifies only the desired targets without requiring extensive hybridization washes, thereby preserving duplex integrity and improving recovery rates.
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 allows for the confident identification of rare mutations with high accuracy and efficiency, minimizing DNA damage and PCR usage, while being cost-effective.
Implementation Method 1
c. extending 5′ adaptors across the exogenous UID sequence and the first segment, thereby generating the complement of the exogenous UID sequence and complement of the first segment
Implementation Method 2
d. covalently linking 3′ end of the complement of the first segment to 5′ ends of the Watson and Crick strands of the double-stranded DNA fragments
Implementation Method 3
wherein the second strand is degradable
Data Source
AI summary
Provided herein are systems, kits, compositions and methods for sequencing library preparation and sequencing workflow (e.g., for the identification of mutations). In certain embodiments, provides herein systems and methods to identically barcode both strands of templates, and PCR-based enrichment of each strand that does not require hybridization capture.