Ligation-Based Nucleic Acid Capture for Ultra-Low Variant Sequencing
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Solution Overview
Problem
Current nucleic acid enrichment methods for next-generation sequencing are time-consuming, labor-intensive, and suffer from low specificity and efficiency, particularly in detecting ultra-low frequency variants and requiring high sequencing capacity due to off-target capture.
Innovation Solution
A ligation-based capture method that enriches for highly fragmented nucleic acids by adding capture moiety modified nucleotides or primers, followed by adapter ligation, capture, and amplification, minimizing adapter carryover and enhancing coverage depth.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If hybridization-based capture assays are used for target enrichment, then sequencing coverage is achieved, but off-target sequence capture occurs and specificity is reduced
Solution Approach 1:
The patent applies local quality by designing capture probes with specific local characteristics - using biotinylated capture probes that hybridize to target sequences with high specificity, while the streptavidin-coated beads provide localized capture functionality. This localized specific interaction minimizes off-target capture while enriching target sequences.
Solution Approach 2:
The patent uses streptavidin-coated magnetic beads as an intermediary between the biotinylated capture probes and the sequencing library. This intermediary system enables specific target enrichment through biotin-streptavidin binding, while allowing for easy separation and washing to remove off-target sequences, thus improving specificity.
2Productivity
If hybridization-based capture assays are used, then target enrichment is achieved, but the process is time-consuming and labor-intensive
Solution Approach 1:
The patent replaces manual, labor-intensive hybridization-based capture procedures with a streamlined magnetic bead-based system. The magnetic beads enable rapid separation and washing steps using magnetic fields, eliminating the need for manual manipulation and significantly reducing processing time while maintaining or improving enrichment efficiency.
Solution Approach 2:
The patent optimizes various parameters including hybridization temperature, bead-to-sample ratio, and washing stringency to achieve rapid and efficient target enrichment. By carefully controlling these parameters, the method achieves high productivity with minimal processing time.
3Measurement precision
If conventional enrichment methods are used, then sequencing is performed, but detection of ultra-low frequency variants is limited
Solution Approach 1:
The patent performs preliminary target enrichment using biotinylated capture probes and streptavidin-coated magnetic beads before sequencing. This preliminary action concentrates ultra-low frequency variants by removing background noise and off-target sequences, enabling their detection with limited sequencing capacity.
Solution Approach 2:
The patent extracts and enriches target sequences containing ultra-low frequency variants from complex genomic backgrounds. By selectively capturing target sequences and removing non-target material, the method enables detection of rare variants that would be undetectable in whole genome sequencing with the same sequencing capacity.
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 unique coverage and specificity, doubling capture efficiency for nucleic acids from tumor or immune-compromised individuals, facilitating accurate detection of ultra-low frequency variants and immune repertoire analysis.
Implementation Method 1
capturing the ligation product by contacting the ligation product with a binding partner of the capture moiety
Data Source
AI summary
Aspects of the technology disclosed herein relate to methods of preparing and analyzing nucleic acids, e.g., cfDNA, and nucleic acids encoding immune receptors and immunoglobulins. In some embodiments, methods for preparing nucleic acids for sequence analysis (e.g., using next-generation sequencing) are provided herein.