Adapter Oligonucleotide for Immune Repertoire Sequencing
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Solution Overview
Problem
Current methods for analyzing nucleic acid variants, particularly in immune receptor loci, face challenges in detecting ultra-low frequency variants and achieving efficient enrichment from complex genomic mixtures, especially in tumor-bearing or immune-compromised individuals.
Innovation Solution
The method involves ligation-based capture using capture moiety modified nucleotides and primers to enrich nucleic acid molecules corresponding to transcribed sequences reflecting recombination and splicing events, improving capture efficiency and depth by incorporating adapter nucleic acids and specific binding partners.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional enrichment methods (multiplex PCR or 5'RACE) are used to enrich rearranged BCRs and TCRs, then the immune repertoire can be analyzed, but the detection sensitivity for ultra-low frequency variants is insufficient
Solution Approach 1:
The patent introduces an adapter oligonucleotide as an intermediary molecule that bridges the target immune receptor sequences and the sequencing platform. This adapter contains a known sequence that facilitates specific capture and enrichment, enabling highly sensitive detection of ultra-low frequency variants while maintaining high enrichment efficiency from complex genomic mixtures
Solution Approach 2:
The method performs preliminary enrichment of target nucleic acid sequences before sequencing by hybridizing capture probes to the adapter-ligated immune receptor sequences. This preliminary capture step concentrates the target variants from complex mixtures, achieving both ultra-sensitive detection and high enrichment efficiency simultaneously
2Measurement precision
If capture efficiency is increased to detect ultra-low frequency variants, then detection sensitivity improves, but the complexity of the enrichment process increases
Solution Approach 1:
The patent merges multiple functions into a single adapter oligonucleotide structure: it contains the hybridization domain for target capture, the known sequence for enrichment, and the platform-specific sequence for sequencing. This consolidation simplifies the overall process while maintaining high detection sensitivity for ultra-low frequency variants
Solution Approach 2:
The adapter oligonucleotide design provides multi-functionality by enabling the same adapter structure to work across different sequencing platforms and for both BCR and TCR repertoire analysis. This universal approach reduces process complexity while achieving ultra-sensitive detection across multiple applications
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 significantly enhances the detection of ultra-low frequency variants, such as splice variants and copy number variants, in immune receptor loci, offering improved sensitivity and specificity for immune repertoire analysis through enhanced front-end capture chemistry.
Implementation Method 1
contacting a nucleic acid molecule comprising a target nucleotide sequence with a capture moiety modified primer that specifically anneals to the target nucleotide sequence under hybridization conditions
Implementation Method 2
ligating an adapter nucleic acid to the double-stranded nucleic acid to produce a ligation product
Implementation Method 3
capturing the ligation product by contacting the ligation product with a binding partner of a capture moiety of the capture moiety modified nucleotide
Data Source
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AI summary
Aspects of the technology disclosed herein relate to methods of preparing and analyzing nucleic acids, e.g., 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.