Immune Repertoire Sequencing With Multiplex Primers and Error Correction
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Solution Overview
Problem
Current methods for analyzing the immune repertoire at high resolution are limited by low throughput and the introduction of sequence errors, making it difficult to effectively capture and understand the diversity of immune cell receptor sequences.
Innovation Solution
A multiplex next-generation sequencing workflow using specific primer sets for V and J gene amplification, followed by error correction, to accurately amplify and sequence immune cell receptor sequences, including TCR and BCR, and generate comprehensive immune repertoire data.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Sanger sequencing is used for immune repertoire analysis, then measurement precision is improved, but productivity deteriorates due to low throughput
Solution Approach 1:
The patent uses next-generation sequencing technology to create multiple copies of immune receptor sequences simultaneously, enabling high-throughput parallel sequencing of thousands of clones. This copying approach allows comprehensive repertoire analysis that maintains sufficient resolution while dramatically increasing throughput compared to traditional Sanger sequencing.
Solution Approach 2:
The patent combines multiple sequencing reads into consensus sequences and merges them into comprehensive repertoire profiles. By merging information from numerous parallel sequencing reactions, the method achieves both high throughput and maintained measurement precision through statistical consolidation of data.
2Productivity
If next generation sequencing is used to capture the repertoire, then productivity is improved, but measurement precision deteriorates due to sequence errors
Solution Approach 1:
The patent implements quality control feedback mechanisms where sequencing errors are detected and corrected through comparison with reference sequences, consensus building, and error rate monitoring. This feedback loop maintains measurement precision by continuously identifying and correcting deviations introduced during high-throughput sequencing.
Solution Approach 2:
The patent employs error correction algorithms and quality filtering procedures that are built into the sequencing workflow beforehand. These pre-planned corrective measures cushion against the introduction of sequence errors by next-generation sequencing, ensuring that accuracy is preserved despite the high-throughput nature of the technology.
3Adaptability or versatility
If multiple primer sets are used for comprehensive V and J gene amplification, then adaptability is improved, but device complexity increases
Solution Approach 1:
The patent designs universal primer sets that can amplify multiple different V and J gene segments simultaneously. These multi-functional primers are engineered to recognize conserved regions across diverse immune receptor genes, allowing a single primer set to serve multiple amplification targets and reducing the overall number of primers needed.
Solution Approach 2:
The patent segments the immune receptor gene loci into distinct V, D, and J regions that can be independently targeted by specific primers. This segmentation allows for systematic coverage of the entire repertoire through coordinated amplification of individual segments, which are then assembled to reconstruct complete receptor sequences.
Data Source
AI summary
The present disclosure provides methods, compositions, kits, and systems useful in the determination and evaluation of the immune repertoire. In one aspect, target-specific primer panels provide for the effective amplification of nucleic acid sequences of murine T cell receptor and/or B cell receptor chains with improved sequencing accuracy and resolution over the repertoire. Variable regions associated with the immune cell receptor are resolved to effectively portray clonal diversity of a biological sample and/or differences associated with the immune cell repertoire of a biological sample.