Multiplexed Digital Quantitation of Lymphoid Receptors
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Solution Overview
Problem
Current methods lack an efficient and accurate way to quantify the absolute abundance of individual T cell or B cell clones and their corresponding nucleic acids in complex mixtures, such as blood samples, which is crucial for understanding immune responses and repertoire diversity.
Innovation Solution
A method involving multiplex PCR to amplify rearranged TCR or IG CDR3 molecules, followed by high-throughput sequencing with unique barcoding, allows for the identification and quantification of individual clones by distributing cells or DNA among multiple wells and using likelihood models to infer clone abundance based on well occupancy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional quantification methods are used for lymphocyte clones in complex mixtures, then the process is simpler, but measurement precision and accuracy are insufficient
Solution Approach 1:
The patent divides the complex mixture of lymphocytes into individual cells through droplet microfluidics, with each droplet containing at most one cell. This segmentation enables independent PCR amplification and sequencing of each clone, achieving precise quantification of individual clones while managing complexity through automated single-cell sorting.
Solution Approach 2:
The patent employs nested barcoding where unique molecular identifiers (UMIs) are incorporated during PCR amplification, and droplet-specific barcodes are added during microfluidics. This nested structure allows tracking of individual clones through multiple processing stages, enabling accurate quantification while maintaining a systematic approach to handle complexity.
2Measurement precision
If high-throughput sequencing with barcoding is used, then measurement precision improves, but device complexity and processing time increase
Solution Approach 1:
The patent performs preliminary actions by pre-forming droplets with template DNA and barcodes before PCR amplification. The droplet microfluidics system pre-separates and labels individual clones with unique identifiers, so that during high-throughput sequencing, the quantification process is already partially complete, reducing overall processing time while maintaining precision.
Solution Approach 2:
The patent changes the physical state of the sample from bulk liquid to discrete droplets, and transforms the information encoding from simple presence/absence to multi-level barcoding with UMIs and droplet IDs. These parameter changes enable parallel processing of thousands of clones simultaneously, improving precision without linearly increasing processing time.
3Productivity
If multiplex PCR amplification is performed, then productivity increases, but measurement precision may deteriorate due to amplification bias
Solution Approach 1:
The patent uses droplet microfluidics to create identical copies of the template DNA in each droplet, with each droplet serving as an independent replication unit. This copying approach allows multiplex PCR to be performed in parallel across thousands of droplets, increasing throughput while maintaining precision because each clone is amplified independently without cross-contamination or competition bias.
Solution Approach 2:
The patent introduces droplet-specific barcodes and UMIs as intermediaries between the original DNA template and the final sequencing data. These intermediaries carry information about the original clone identity and allow accurate tracking through the multiplex PCR process, enabling productivity increase while correcting for any amplification bias that occurs.
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 accurate estimation of the absolute abundance of T cell or B cell clones, reducing uncertainty and providing a comprehensive understanding of immune receptor diversity in complex samples.
Implementation Method 1
A method involving multiplex PCR to amplify rearranged TCR or IG CDR3 molecules
Implementation Method 2
followed by high-throughput sequencing with unique barcoding, allows for the identification and quantification of individual clones
Data Source
AI summary
The invention relates to methods and compositions for estimating the absolute abundance individually for each unique rearranged lymphocyte receptor in a mixed sample.