Oligonucleotide Adapter Attachment for Liquid Biopsy
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Solution Overview
Problem
Current methodologies for obtaining and processing source samples fail to fully leverage the power and sensitivity of next-generation sequencing platforms to accurately detect rare sequences, particularly in cancer diagnosis, where the fraction of tumor-derived cfDNA is very low, leading to biases and loss of sequence information.
Innovation Solution
A method involving the attachment of an oligonucleotide adapter to the 3' end of double-stranded DNA fragments, followed by linear amplification, hybridization with a target-specific oligonucleotide probe, purification, and PCR amplification to produce sequencing template molecules, which enhances the conversion efficiency and reduces signal bias.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional adapter attachment methods are used, then the process is simple, but the conversion efficiency is low and signal bias occurs
Solution Approach 1:
The adapter attachment process is divided into two distinct stages: first, attaching a partial adapter to the DNA fragment, and second, completing the adapter attachment through probe hybridization and extension. This segmentation allows each stage to be optimized independently, improving overall conversion efficiency while maintaining manageable process complexity.
Solution Approach 2:
The method performs preliminary adapter attachment to DNA fragments before complete adapter ligation. By pre-attaching the partial adapter and then using probe-driven completion, the system ensures that even fragments with suboptimal end structures can be successfully adapted, reducing signal bias and improving reliability.
2Measurement precision
If standard PCR amplification is used, then the amplification is fast, but rare sequences are lost due to bias
Solution Approach 1:
The patent introduces an oligonucleotide probe as an intermediary between the partially adapted DNA fragment and the complete adapter. This probe serves as a mediator that facilitates accurate and biased-free amplification of rare sequences, improving detection accuracy while maintaining amplification efficiency through the bridge it creates.
3Reliability
If adapter attachment is performed on all DNA fragments, then coverage is comprehensive, but fragments with poor end structure fail to adapt and are lost
Solution Approach 1:
Instead of requiring complete adapter attachment for all fragments in a single step, the method performs partial adapter attachment first, then uses probe hybridization to complete the process for fragments that need it. This partial action approach ensures that fragments with poor end structures still have a chance to be recovered, improving both reliability and quantity of recovered fragments.
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 improves the sensitivity and accuracy of detecting rare sequences by ensuring the attachment of adapters to DNA fragments, even if one end is not properly adapted, and reduces the loss of sequence information, enabling more reliable detection of tumor-derived DNA in liquid biopsies.
Implementation Method 1
hybridizing the at least one complementary strand with an oligonucleotide probe, wherein the oligonucleotide probe comprises a hybridization domain with a sequence that hybridizes to a target sequence in the complement strand
Data Source
AI summary
The disclosure provides methods and reagents for preparing DNA libraries from biological materials for targeted sequencing. The approach can enhance the efficiency and sensitivity of targeted sequencing applications, such as liquid biopsy analyses to assess genetically driven conditions. In an embodiment, the disclosed method comprises attaching the 5′ end of an oligonucleotide adapter to the 3′ end of double-stranded DNA fragment to produce an adapter/fragment chimeric molecule; producing at least one complementary strand of the adapter/fragment chimeric molecule by linear amplification; hybridizing the at least one complementary strand with an oligonucleotide probe, wherein the oligonucleotide probe comprises a hybridization domain with a sequence that hybridizes to a target sequence in the complement strand to produce a targeted complement strand/probe duplex; purifying the targeted complement strand/probe duplex; and extending the probe in the purified targeted complement strand/probe duplex and amplifying with PCR to produce a plurality of sequencing template molecules.