TCR Recombination Sequencing With Universal Adapters and Nested PCR
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for analyzing the T-cell receptor (TCR) repertoire face significant limitations, including PCR primer biases, low efficiency, and difficulty in discovering novel elements or mutations, making it challenging to exhaustively sequence and analyze the TCR repertoire for clinical applications.
Innovation Solution
A method called T-seq, which involves ligating universal primers to fragmented mRNA and performing nested PCR with 3′-oligos from the constant C region and a universal 5′ adapter, allowing for unbiased and efficient sampling of recombination junctions, particularly the CDR3 region, with high read coverage and reduced sequencing costs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional PCR methods are used to sequence TCR repertoire, then the process is simple, but primer biases and low efficiency reduce measurement precision and reliability
Solution Approach 1:
The patent introduces universal adapters as intermediary elements that ligate to the 5' ends of TCR cDNA fragments. These adapters serve as mediators between the cDNA fragments and PCR primers, eliminating direct primer binding to the TCR sequence. This intermediary approach removes primer bias while maintaining amplification efficiency, directly resolving the contradiction between measurement precision and reliability.
Solution Approach 2:
The patent segments the TCR repertoire sequencing process into distinct steps: fragmenting the TCR cDNA, ligating universal adapters to fragments, and then performing PCR amplification. This segmentation separates the adapter ligation step from the amplification step, allowing for unbiased representation of all TCR junctions while maintaining high efficiency through optimized PCR conditions.
2Measurement precision
If exhaustive sequencing of TCR repertoire is performed, then measurement precision improves, but sequencing costs and time increase
Solution Approach 1:
The patent employs universal adapters that can be ligated to any TCR cDNA fragment regardless of its specific sequence or junction type. This universal adapter design enables a single amplification protocol to efficiently sequence the entire TCR repertoire, including novel elements and mutations, without requiring multiple specialized reactions. This multi-functionality achieves comprehensive sequencing coverage while reducing time and cost.
Solution Approach 2:
The patent changes the key parameter of using universal adapters instead of sequence-specific primers. This parameter change enables the system to handle diverse TCR junctions uniformly, achieving exhaustive sequencing coverage. The universal adapter approach maintains high efficiency and reduces sequencing costs while providing complete repertoire analysis, resolving the contradiction between measurement precision and time loss.
3Measurement precision
If novel elements and mutations are discovered through sequencing, then measurement precision improves, but difficulty in detecting and measuring increases
Solution Approach 1:
The universal adapters act as intermediaries that uniformly represent all TCR fragments, including those containing novel elements or mutations. By using these adapters, the sequencing process captures diverse junctions without bias, and the standardized adapter sequence simplifies data analysis by providing a consistent reference point for identifying novel segments and mutations.
Solution Approach 2:
The patent creates multiple copies of TCR junctions through PCR amplification using universal adapters. This copying process generates sufficient read depth to detect rare novel elements and mutations while maintaining a standardized framework for analysis. The replicated sequences facilitate easier detection and measurement of diverse TCR junctions compared to direct single-pass sequencing.
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
T-seq provides a cost-effective, accurate, and rapid method for profiling the TCR repertoire, enabling the discovery of novel segments and reducing sequencing costs, thereby improving the analysis of TCR diversity for clinical applications.
Implementation Method 1
The first adaptor nucleic acids include a first hybridization region having a first predefined hybridization sequence
Implementation Method 2
selectively amplifying respective ligated nucleic acid fragments, in the plurality of ligated nucleic acid fragments, containing a recombined junction
Data Source
AI summary
The present disclosure relates to methods for determining recombination diversity at a genomic locus of interest. The method includes fragmenting nucleic acids isolated from immune cells, ligating adaptors to the fragmented or amplified nucleic acids, and selectively amplifying nucleic acids containing a recombined junction at the genomic locus of interest. Selective amplification is achieved by using a first primer that hybridizes to an adaptor sequence and a second primer that hybridizes at a constant region downstream of the recombined junction. The selectively amplified nucleic acids may be sequences and analyzed to determine recombination diversity at the genomic locus.