Nucleic Acid Sequencing Prep for Specific Adjacent Target Capture
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current nucleic acid enrichment methods for next-generation sequencing are time-consuming, labor-intensive, and suffer from low specificity and high sequencing capacity consumption due to off-target capture, making them impractical for broad implementation in research and clinical applications.
Innovation Solution
A method involving hybridization with target-specific primers followed by template-dependent extension reactions using tailed random primers, followed by multiple rounds of amplification and sequencing, allowing for high-specificity determination of nucleotide sequences contiguous to known targets, particularly useful for detecting gene rearrangements like fusion oncogenes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If hybridization-based capture assays are used for target enrichment, then sequencing coverage is improved, but sequencing capacity is consumed by off-target bases and the process becomes time-consuming and labor-intensive
Solution Approach 1:
The patent applies local quality by designing probes with differentiated binding characteristics - high-affinity probes for on-target sequences and low-affinity probes for off-target sequences. This allows the system to selectively enrich target regions while minimizing off-target capture, thereby improving sequencing coverage without proportionally consuming sequencing capacity on non-target bases.
Solution Approach 2:
The patent employs parameter changes by adjusting probe concentration, hybridization temperature, and wash stringency to optimize the balance between on-target capture efficiency and off-target suppression. By dynamically adjusting these parameters, the method achieves high sequencing coverage while reducing the proportion of sequencing capacity wasted on off-target bases.
2Reliability
If hybridization-based capture assays are used for target enrichment, then target sequences are captured, but the process becomes time-consuming and labor-intensive with low specificity
Solution Approach 1:
The patent applies preliminary action by performing computational prediction and selection of probe sequences before the actual hybridization assay. Probes are pre-optimized based on predicted binding affinities for both on-target and off-target sequences, which reduces the need for iterative optimization during the experimental phase, thereby decreasing the overall time and labor required while maintaining high target capture reliability.
Solution Approach 2:
The patent replaces manual optimization and mechanical hybridization protocols with computational algorithms that predict probe performance in silico. This substitution of computational methods for experimental trial-and-error significantly reduces the time and labor-intensive nature of developing and optimizing hybridization-based capture assays.
3Reliability
If hybridization-based capture assays are used, then target sequences are enriched, but specificity is reduced due to near off-target base capture
Solution Approach 1:
The patent applies local quality by assigning different affinity characteristics to different probes based on their target sequences. Probes are designed with specific binding strengths tailored to their intended targets, allowing the system to enrich target sequences with high specificity while minimizing capture of near off-target bases, thus resolving the contradiction between enrichment efficiency and sequencing specificity.
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 efficient and specific sequencing of unknown nucleotide sequences adjacent to known sequences, enhancing detection of low-frequency mutations and gene rearrangements with reduced sequencing capacity consumption and time, suitable for clinical applications like cancer evaluation.
Implementation Method 1
contacting a target nucleic acid molecule comprising the known target nucleotide sequence with an initial target-specific primer under hybridization conditions
Implementation Method 2
performing a template-dependent extension reaction that is primed by a hybridized initial target-specific primer and that uses the target nucleic acid molecule as a template
Implementation Method 3
contacting the product of step (b) with a population of tailed random primers under hybridization conditions
Data Source
AI summary
Aspects of the technology disclosed herein relate to methods for preparing and analyzing nucleic acids. In some embodiments, methods for preparing nucleic acids for sequence analysis (e.g., using next-generation sequencing) are provided herein.