Single-Cell TCR Sequencing via Multiplexed Nested PCR
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Solution Overview
Problem
Current methods for analyzing T cell receptor (TCR) repertoires are limited in throughput and often require artificial cloning procedures, which can skew the in vivo state, making it difficult to understand TCR usage at the single-cell level and the nature of immune responses.
Innovation Solution
A multiplex panel of chimeric primer sequences is developed to amplify TCRαβ sequences from single cells using next-generation sequencing, allowing for high-throughput analysis of TCRαβ sequences from up to 6300 cells simultaneously through a nested PCR approach with barcoded primers for cell identification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If single-cell based methods are used to sequence TCR repertoire, then direct pairing of α and β chains is permitted, but throughput is limited and processing time is increased
Solution Approach 1:
The patent divides the TCR sequencing process into distinct segments: (1) enrichment of specific TCR sequences using tetramer-based magnetic beads, (2) single-cell isolation and lysis, (3) targeted amplification of TCRα and TCRβ chains using separate primer sets, and (4) paired-end sequencing. This segmentation allows each step to be optimized independently, achieving both high precision in TCR pairing and improved throughput by processing multiple cells in parallel through the enriched pool approach
Solution Approach 2:
The patent performs preliminary enrichment of TCR sequences of interest using tetramer-based magnetic beads before single-cell isolation. This preliminary action concentrates the rare epitope-specific T cells from the entire population, so that subsequent single-cell processing focuses only on relevant cells. This pre-enrichment step dramatically improves throughput by avoiding the need to sequence entire T cell populations while maintaining the ability to directly pair α and β chains at single-cell resolution
2Productivity
If deep sequencing-based methods amplifying single chains from pools are used, then throughput is increased, but pairing of α and β chains requires complex sort conditions and algorithmic imputation
Solution Approach 1:
The patent uses single-cell isolation as an intermediary step between pool enrichment and sequencing. By isolating individual cells after tetramer enrichment and lysing them in separate reactions, the patent creates a physical link between TCRα and TCRβ chains from the same cell. This intermediary approach simplifies pairing compared to algorithmic methods because the physical proximity in single-cell lysates provides direct evidence of pairing without requiring complex computational imputation
Solution Approach 2:
The patent performs separate amplifications of TCRα and TCRβ chains in the same reaction well, creating copies of both chains from the same single cell. These amplified products are then sequenced together, providing direct experimental evidence of pairing. This copying approach in a shared reaction space simplifies the pairing process compared to analyzing separate pools, as the co-presence of both chain amplification products in the same well confirms they originated from the same cell
3Quantity of substance
If artificial cloning procedures are used for TCR analysis, then TCR sequences can be obtained, but the in vivo state is skewed and natural TCR usage is distorted
Solution Approach 1:
The patent replaces the mechanical cloning process (physical manipulation of cells through multiple isolation and culture steps) with a direct molecular approach: extracting RNA from fixed single cells, converting to cDNA, and performing PCR amplification. This substitution eliminates the selective pressures and artificial conditions inherent in cloning procedures, preserving the natural abundance and diversity of TCR sequences as they exist in vivo while still obtaining sufficient quantities for sequencing analysis
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 method enables efficient, unbiased, and cell-number-agnostic analysis of TCRαβ sequences from individual T cells, facilitating the understanding of immune responses and potential applications in diagnosing and treating diseases such as inflammatory disorders, autoimmune diseases, and cancer.
Implementation Method 1
amplifying nucleic acid molecules encoding TCR α and β from one or more single T cells using the first set of primers from the kit to produce a first set of amplicon products
Data Source
AI summary
A kit and method for analyzing nucleic acid molecules encoding T cell receptor (TCR) a and β from individual T cells are disclosed. In particular, a method for analyzing individual T cells using high-throughput multiplex amplification and deep sequencing of nucleic acids encoding TCRαβ is provided.