Immune Repertoire Sequencing via 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 in next-generation sequencing, making it difficult to effectively capture and understand the complex populations of immune cell receptor sequences.
Innovation Solution
A multiplex next-generation sequencing workflow using specific primer sets targeting variable, diversity, and joining gene segments of immune receptors, along with error correction methods to generate accurate and comprehensive sequence information.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If next-generation sequencing is used to capture the immune repertoire, then throughput and coverage are improved, but sequence errors are introduced reducing accuracy
Solution Approach 1:
The patent applies preliminary action by performing error correction through clustering and consensus building before final sequence identification. Multiple reads are processed in advance to identify and correct errors, ensuring high accuracy is achieved prior to repertoire characterization, thus resolving the contradiction between high throughput and sequence accuracy.
Solution Approach 2:
The patent implements feedback mechanisms through iterative clustering and consensus building processes. Sequencing reads are clustered based on similarity, and consensus sequences are generated by comparing multiple reads within each cluster. This feedback loop allows errors to be identified and corrected based on the majority signal, maintaining high accuracy while processing large throughput data.
2Measurement precision
If traditional Sanger sequencing is used, then sequence accuracy is maintained, but throughput is limited reducing repertoire coverage
Solution Approach 1:
The patent merges the advantages of both Sanger sequencing and NGS by combining high-throughput NGS data generation with Sanger-like accuracy through clustering and consensus building. Multiple NGS reads are combined and processed to generate consensus sequences that achieve accuracy comparable to Sanger sequencing while maintaining the high throughput capability of NGS platforms.
3Adaptability or versatility
If multiplex amplification with multiple primer sets is performed, then comprehensive repertoire capture is improved, but complexity of the workflow increases
Solution Approach 1:
The patent applies universality by designing a multiplex amplification system where a single reaction mixture containing multiple primer sets can simultaneously amplify multiple different immune receptor variable regions. The universal PCR conditions and single-tube format allow comprehensive repertoire capture without requiring separate reactions for each target, thus reducing workflow complexity while maintaining versatility.
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 enables high-accuracy amplification and sequencing of immune cell receptor sequences, improving the detection and characterization of immune repertoires by reducing false positives and enhancing read assignment accuracy.
Implementation Method 1
a plurality of variable (V) gene primers directed to a majority of different variable regions of an immune receptor coding sequence
Implementation Method 2
amplification and sequencing of immune cell 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 sequences of 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.