Probe Extension for AT-Rich Sequence Enrichment
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Solution Overview
Problem
Current methods for preparing cell-free nucleic acid sequencing libraries often result in the loss of AT-rich sequences and carry over non-target nucleic acids due to low probe-target hybridization and insufficient washing, leading to a reduction in on-target sequences.
Innovation Solution
A method involving the use of capture probes complementary to target nucleic acids, hybridization, extension with a polymerase, and isolation of extended probe-target constructs to enrich sequencing libraries, particularly for AT-rich sequences, improving the efficiency of targeted sequencing library preparation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If conventional hybridization-based enrichment is used, then sequencing library preparation is simplified, but AT-rich sequences are lost due to low probe-target hybridization
Solution Approach 1:
The patent applies preliminary action by extending the capture probes with a polymerase before the washing step. This extension creates longer probe-target hybrids that are more stable during subsequent washing, preventing AT-rich sequence loss while maintaining the simplicity of the enrichment approach
Solution Approach 2:
The patent changes the physical parameter of probe length by extending short capture probes into longer constructs using polymerase. This parameter change increases hybridization stability and prevents loss of AT-rich sequences during washing while maintaining ease of library preparation
2Reliability
If insufficient washing is performed, then probe-target hybridization is maintained, but non-target nucleic acids are carried over
Solution Approach 1:
The patent performs probe extension as a preliminary action before washing. The extended probes create more stable hybrids that can withstand stringent washing conditions, allowing thorough removal of non-target nucleic acids while maintaining probe-target hybridization stability
Solution Approach 2:
The probe extension acts as a cushioning measure beforehand, creating extended probe-target constructs that are more resistant to dissociation during washing. This protects the specific hybrids from being lost while allowing non-specific bindings to be removed
3Manufacturing precision
If extensive washing is performed to remove non-target nucleic acids, then enrichment purity is improved, but AT-rich sequences are lost due to low hybridization stability
Solution Approach 1:
By extending the probes before washing, the patent creates stable hybrids that can endure extensive washing. This preliminary extension ensures that even AT-rich sequences with lower inherent stability remain bound during thorough washing, achieving both high purity and minimal loss
Solution Approach 2:
The patent changes the hybridization parameter by extending probe length, which increases melting temperature and stability. This allows extensive washing to be performed without losing AT-rich sequences, simultaneously improving enrichment purity and preventing sequence loss
4Productivity
If short capture probes are used, then library preparation efficiency is improved, but hybridization stability is reduced leading to sequence loss
Solution Approach 1:
The patent uses short capture probes for efficient library preparation, then performs preliminary extension with polymerase to create stable hybrids. This two-step approach maintains the productivity benefits of short probes while achieving the stability of long probes through the extension step
Solution Approach 2:
The patent makes the probe dynamic by transforming it from a static short oligonucleotide into an extended construct. The extension step allows the probe to adapt its length in situ, combining the efficiency of short probes with the stability of long probes
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 the enrichment of AT-rich sequences and reduces non-target binding, resulting in improved sequencing library quality and reduced sequencing depth requirements for cancer detection and monitoring.
Implementation Method 1
extending the capture probes using a polymerase and the target nucleic acids as a template to generate a plurality of extended probe-target nucleic acid constructs
Implementation Method 2
hybridizing the plurality of capture probes to the one or more target nucleic acids
Data Source
AI summary
Aspects of the invention include methods for preparing an enriched sequencing library. In some embodiments, the methods involve preparing a sequencing library that is enriched for AT-rich sequences. In certain embodiments, the methods involve determining a presence or an absence of cancer, determining a cancer stage, monitoring cancer progression, and/or determining a cancer classification in a subject by analyzing an enriched sequencing library.