Immune Repertoire Sequencing Multiplex Amplification 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 during next-generation sequencing, making it difficult to accurately capture the true diversity of immune cell receptor sequences.
Innovation Solution
A multiplex next-generation sequencing workflow that involves multiplex amplification of immune receptor genomic DNA sequences using sets of V and J gene primers, followed by error correction strategies to generate comprehensive and accurate 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 multiplex amplification with error correction before sequencing. Multiple primer sets targeting different V and J gene segments are used to amplify immune receptor sequences in advance, allowing error correction to be applied to the amplified products before they undergo sequencing. This preliminary error correction step prevents sequencing errors from compromising the accuracy of the final repertoire analysis while maintaining the high throughput capability of NGS.
2Measurement precision
If Sanger sequencing is used for high resolution analysis, then sequence accuracy is improved, but throughput is limited reducing efficiency
Solution Approach 1:
The patent applies segmentation by dividing the immune repertoire analysis into multiple targeted amplification reactions. Instead of attempting to sequence all immune receptor sequences in a single Sanger reaction, the method uses multiple primer sets that each target specific V and J gene segment combinations. Each amplification reaction focuses on a manageable subset of sequences, enabling accurate Sanger sequencing of individual clones while collectively covering the entire repertoire through the multiplexed approach.
3Adaptability or versatility
If multiplex amplification is performed to capture diverse immune receptor sequences, then repertoire coverage is improved, but amplification bias and errors increase
Solution Approach 1:
The patent applies local quality by using different primer sets with optimized characteristics for different regions of the immune receptor genes. Each primer set is specifically designed to target particular V and J gene segments with appropriate annealing temperatures and binding affinities. This localized optimization ensures that each amplification reaction is tailored to its specific target region, minimizing amplification bias within each reaction while collectively achieving comprehensive repertoire coverage across all gene segments.
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 receptor gene sequences, improving the resolution and accuracy of immune repertoire analysis, and facilitating better understanding of immune responses and diagnostic capabilities.
Implementation Method 1
amplification comprising performing a multiplex amplification reaction in the presence of a polymerase under amplification conditions to produce a plurality of amplified target sequences
Data Source
AI summary
The present disclosure provides methods, compositions, kits, and systems useful in the determination and evaluation of the immune repertoire using genomic DNA from a biological sample. 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. Nucleic acid sequences of variable regions associated with the immune cell receptor are determined to effectively portray clonal diversity of a biological sample and/or differences associated with the immune cell repertoire of a biological sample.