Universal Tail-Adaptor Ligation for Target Enrichment
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Solution Overview
Problem
Current methods for target enrichment prior to next-generation sequencing are costly, time-consuming, labor-intensive, and suffer from low specificity, particularly for detecting genomic rearrangements with unknown fusion partners, and require the use of both forward and reverse primers, which is not applicable for all sequencing approaches.
Innovation Solution
A method involving ligation of a target nucleic acid with a universal oligonucleotide tail-adaptor, followed by amplification using specific primers and subsequent sequencing, which allows for the determination of nucleotide sequences contiguous to a known target sequence with high specificity and sensitivity, enabling the detection of gene rearrangements like ALK, ROS1, and RET without requiring known fusion partners.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If hybridization-based capture assays are used for target enrichment, then coverage depth and dynamic range are improved, but time consumption and labor intensity increase
Solution Approach 1:
The patent replaces the mechanical/hybridization-based capture system with a PCR amplification system. Instead of using hybridization probes to enrich targets, the method uses PCR primers to specifically amplify target sequences, achieving the same enrichment effect through a different mechanism that is faster and less labor-intensive
Solution Approach 2:
The patent changes the fundamental parameter of the enrichment mechanism from hybridization-based to PCR-based. This parameter change transforms the process into a more efficient system that maintains high coverage depth while reducing time consumption and labor requirements
2Measurement precision
If hybridization-based capture assays are used for target enrichment, then coverage depth is improved, but specificity decreases
Solution Approach 1:
The patent replaces the hybridization-based system with PCR amplification, which inherently provides higher specificity. PCR primers can be designed to match specific target sequences with high precision, eliminating the off-target binding issues inherent in hybridization-based approaches
Solution Approach 2:
The patent applies local quality by designing primers with specific binding regions that target only the desired sequences. The primers are carefully designed to have high complementarity to the target loci while minimizing similarity to non-target sequences, thereby achieving high specificity in the amplified regions
3Productivity
If conventional PCR design with forward and reverse primers is used, then amplification is achieved, but adaptability for unknown fusion partners decreases
Solution Approach 1:
The patent segments the primer design into two functional parts: a target-specific portion that binds to known sequences and a universal portion that can pair with various fusion partners. This segmentation allows the assay to maintain high productivity for known targets while gaining versatility for unknown fusion partners
Solution Approach 2:
The patent implements universality by incorporating universal primers that can bind to multiple different fusion partner sequences. The universal portion of the primers is designed to be complementary to a wide range of potential fusion partners, enabling the same primer set to detect both known and unknown fusion partners in cancer samples
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 method enhances the specificity and efficiency of target enrichment, allowing for the detection of gene rearrangements with high accuracy and sensitivity, facilitating the identification of oncogene rearrangements for personalized cancer treatment by determining the presence of ALK, ROS1, or RET rearrangements in tumor samples.
Implementation Method 1
ligating a target nucleic acid comprising the known target nucleotide sequence with a universal oligonucleotide tail-adaptor
Implementation Method 2
the first target-specific primer comprises a nucleic acid sequence that can specifically anneal to the known target nucleotide sequence of the target nucleic acid at the annealing temperature
Data Source
AI summary
The technology described herein is directed to methods of determining oligonucleotide sequences, e.g. by enriching target sequences prior to sequencing the sequences.