Linked Transcript Sequencing Immune Repertoires

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Solution Overview

Problem

Current sequencing technologies face challenges in accurately determining the B-cell and T-cell receptor repertoires due to the complexity of determining the sequences of two independent polynucleotides, which is essential for immune function recognition and analysis.

Innovation Solution

A method involving the hybridization of bridge oligonucleotides and interposing oligonucleotide probes to form bridged polynucleotide complexes, followed by extension and ligation to create integrated strands, which are then amplified to produce tagged complements of two independent single-stranded polynucleotides, enabling precise sequencing of immune receptor repertoires.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If conventional sequencing technologies are used to determine B-cell and T-cell receptor repertoires, then the sequencing process can be performed with existing methods, but the accuracy and precision of determining two independent polynucleotide sequences are insufficient

Engineering Contradiction:

Improvesequencing accuracyVSAvoidmethod complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The method segments the complex task of determining two independent polynucleotide sequences into distinct functional modules: bridge oligonucleotide hybridization to link the two chains, interposing oligonucleotide probe hybridization to capture specific regions, extension to synthesize complementary strands, and ligation to covalently join fragments. This segmentation allows each step to be optimized independently while maintaining overall sequencing accuracy.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention introduces several intermediary oligonucleotides that facilitate the sequencing process: bridge oligonucleotides that hybridize to both polynucleotides of interest, interposing oligonucleotide probes that contain barcode sequences and primer binding sites, and extension primers. These intermediaries mediate the complex interactions between the two independent polynucleotides, enabling accurate determination of their sequences without direct complex interaction between them.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Measurement precision

If high-precision sequencing of immune receptor repertoires is achieved through complex hybridization and ligation processes, then sequencing accuracy is improved, but the process time and procedural steps increase

Engineering Contradiction:

Improverepertoire sequencing precisionVSAvoidsequencing process time

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The method performs preliminary hybridization of bridge oligonucleotides to both polynucleotides before the actual sequencing steps. This preliminary action establishes the correct spatial arrangement and brings the two independent sequences into proximity, so that subsequent probe hybridization and extension can proceed efficiently in a coordinated manner, reducing overall process time.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The invention merges multiple functions into the interposing oligonucleotide probes: they contain hybridization sequences for binding to target polynucleotides, barcode sequences for identification, and primer binding sequences for extension. This merging allows a single oligonucleotide to perform multiple functions simultaneously, reducing the number of separate steps required and decreasing total process time.

Inventive Principle:

Principle #5Merging (Combining)

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 allows for high-precision sequencing of immune receptor repertoires, facilitating the study of tumor-infiltrating lymphocytes and vaccine responses by overcoming the limitations of existing sequencing technologies.

Implementation Method 1

hybridizing a bridge oligonucleotide to a first polynucleotide and a second polynucleotide, thereby forming a bridged polynucleotide complex

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

hybridizing one or more interposing oligonucleotide probes to the first polynucleotide and second polynucleotide

Methodology Applied

Scientific EffectHybridization:

Implementation Method 3

extending the 3' end of each second hybridization sequence of the interposing oligonucleotide probes and the 3' end of the hybridization sequence of the bridge oligonucleotide with one or more polymerases

Methodology Applied

Scientific EffectPolymerization: