Liquid-Phase Hybrid Capture with Tiled Short Probes for Small Panels
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Solution Overview
Problem
Current liquid-phase hybrid capture technologies face challenges with low capture efficiency and on-target rates, particularly for small target regions, lengthy operation times, and complex, cumbersome processes, which are unsuitable for automated workstations and high-throughput sequencing applications.
Innovation Solution
A method for designing probes with complementary paired segments and optimized annealing temperatures, allowing for high recovery efficiency and uniformity, combined with a streamlined hybrid capture process that reduces operation time and simplifies automation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional probes of 120 nt are used for hybrid capture, then the probe binding ability and specificity are optimized, but the capture efficiency and on-target rate are low for small target regions
Solution Approach 1:
The patent divides the conventional 120 nt probe into multiple shorter probes (e.g., 3-5 probes of 20-40 nt each) that tile across the target region. This segmentation allows each short probe to bind effectively to small target fragments (100-400 bp) while collectively covering the entire target region, thereby improving capture efficiency without sacrificing binding specificity
Solution Approach 2:
The patent transitions from using single long probes to using multiple short probes arranged in a tiled pattern across the target region. This dimensional change from one-dimensional long probe binding to two-dimensional tiled coverage increases the probability of probe-target hybridization and improves on-target rate for small regions
2Reliability
If the probe sequence length is increased to improve binding ability, then the hybridization annealing temperature is appropriate, but the synthesis difficulty and quality control become more challenging
Solution Approach 1:
The patent changes the probe length parameter from conventional 120 nt to shorter lengths of 20-40 nt. This parameter change makes probe synthesis easier and quality control more reliable, while the tiled arrangement of multiple short probes compensates for the reduced individual binding strength, maintaining overall capture efficiency
3Reliability
If long insert fragments are used for sequencing library, then the probe binding capacity increases, but the middle part cannot be read and PCR amplification becomes challenging
Solution Approach 1:
The patent segments the target region into multiple short probe binding sites along the insert fragment. This segmentation allows multiple short probes to bind at different locations on the same fragment, increasing total binding capacity without requiring excessively long insert fragments, thereby maintaining PCR amplification efficiency and sequencing readability
4Reliability
If the hybrid capture process is optimized for high capture efficiency, then the on-target rate improves, but the operation time becomes lengthy and the process becomes complex
Solution Approach 1:
The patent changes the hybridization temperature parameter to a higher range (50-65°C, preferably 60-65°C) which accelerates hybridization kinetics and reduces operation time. The use of multiple short probes with this elevated temperature parameter achieves high on-target rate while reducing the capture process to 1-2 hours
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
The method achieves high capture efficiency, improved binding ability, and uniformity, meeting the requirements for automated workstations and reducing the overall process time from days to hours while maintaining high on-target rates.
Implementation Method 1
a biotin-labeled probe specifically binds to a target region in a solution
Implementation Method 2
target fragments captured by the probe are enriched by streptavidin magnetic beads
Data Source
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AI summary
The present disclosure provides a liquid-phase hybrid capture method, including the steps of probe design and synthesis, library construction and hybrid capture. The hybrid capture is optimized, and the method of the present disclosure can be used for the construction of various nucleic acid sample capture libraries from different sources, effectively increasing the capture efficiency, shortening the experimental time, and simplifying the experimental process, and the method is used for target region (Panel) capture.