Duplex Sequencing Library Preparation for Rare Mutation Detection
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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 anchored PCR enrichment and in silico reconstruction to identify mutations present on both strands, minimizing DNA damage and PCR requirements.
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 patent applies preliminary action by performing molecular barcoding during library preparation, before sequencing occurs. Unique molecular identifiers (UMIs) are attached to each DNA molecule in advance, allowing subsequent error correction by comparing multiple sequencing reads of the same original molecule. This preliminary tagging enables confident detection of rare mutations even when sequencing errors are present.
Solution Approach 2:
The patent uses copying by generating multiple redundant sequencing reads of each barcoded DNA molecule. By sequencing the same original template molecule multiple times (creating copies of the sequence data), the true mutation signal can be distinguished from random sequencing errors through consensus analysis of the replicated reads.
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
Solution Approach 1:
The patent applies segmentation by separating the barcoding process into distinct stages: first attaching UMIs to single-stranded DNA, then using strand-displacement synthesis to create duplex molecules. This segmentation allows optimization of each step independently, improving overall duplex conversion efficiency while maintaining accurate molecular barcoding for mutation detection.
Solution Approach 2:
The patent uses an intermediary mechanism by employing strand-displacement synthesase as a mediator enzyme to facilitate the conversion of single-stranded barcoded DNA to duplex molecules. This intermediary enzyme enables efficient and specific duplex formation without requiring complex additional reagents or conditions.
3Quantity of substance
If hybridization capture is used to enrich libraries, 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 patent applies taking out by removing the problematic hybridization capture step from the workflow and replacing it with PCR-based enrichment. This extraction of the flawed method eliminates the issue of poor duplex recovery while maintaining the ability to enrich target regions, particularly for small targets where hybridization capture fails to scale effectively.
Solution Approach 2:
The patent changes the enrichment parameter from hybridization-based capture to PCR amplification. This parameter change allows for better control of enrichment specificity and efficiency, particularly for small target regions, while preserving duplex molecule integrity and achieving higher duplex recovery rates.
4Productivity
If CRISPR-DS is used to improve duplex recovery, then recovery can reach up to 15%, but it is not applicable to cell-free DNA
Solution Approach 1:
The patent applies universality by developing a PCR-based enrichment approach that works across multiple sample types including cell-free DNA, formalin-fixed paraffin-embedded (FFPE) samples, and fresh frozen tissues. This universal method achieves high duplex recovery rates (up to 80% in some cases) without being limited to specific sample types, unlike CRISPR-DS which only works with certain DNA preparations.
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
Enables accurate and efficient detection of rare mutations with high confidence, even in limited DNA samples, by ensuring mutations are identified on both strands, thus reducing artifacts and sequencing errors.
Implementation Method 1
performing a nick translation-like reaction to extend the 5' adaptors across the exogenous UID sequence of the 3' adaptors
Implementation Method 2
covalently linking the extended 5' adaptor to the 5' ends of the Watson and Crick strands of the double-stranded DNA fragments
Implementation Method 3
amplifying the adaptor-ligated, double-stranded DNA fragments to produce amplicons
Implementation Method 4
degrading the degradable 3' blocking group
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.