Synthetic Randomer Molecules for PCR Amplification Bias Correction
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Solution Overview
Problem
Current methods for quantifying adaptive immune cells in complex biological samples face challenges in accurately determining absolute numbers and correcting for amplification bias in multiplex PCR, requiring extrinsic data and unable to provide absolute quantitation.
Innovation Solution
The use of synthetic molecules with unique barcodes and random oligonucleotide sequences in PCR reactions allows for precise measurement of amplification bias correction and absolute input template quantitation without the need for extrinsic data, by clustering and normalizing sequencing reads to accurately represent the number of input biological sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current methods for quantifying adaptive immune cells are used, then the process can be performed with standard PCR techniques, but the accuracy of absolute quantitation is insufficient due to inability to correct for amplification bias
Solution Approach 1:
The patent introduces synthetic template molecules as intermediary control elements that mediate between the PCR amplification process and the quantitation measurement. These synthetic templates with known concentrations and unique barcodes serve as internal references that allow correction of amplification bias, enabling accurate absolute quantitation of adaptive immune cells without requiring complex external calibration systems
Solution Approach 2:
The patent changes the parameter of template diversity by incorporating synthetic templates with numerous unique barcode sequences. This parameter change allows the system to track and correct for amplification bias across multiple parallel reactions, transforming the quantitation method from relative to absolute while maintaining practical complexity through standardized protocols
2Reliability
If extrinsic data is used for amplification bias correction, then some correction can be achieved, but the reliability is reduced due to dependence on external reference data
Solution Approach 1:
The patent implements self-service by incorporating internal control mechanisms within the PCR reaction itself. The synthetic template molecules with known concentrations serve as self-contained references that allow the reaction system to automatically correct for its own amplification bias without requiring external calibration data or reference measurements, thereby improving reliability while eliminating dependence on extrinsic information
Solution Approach 2:
The patent establishes a feedback loop where the known concentrations of synthetic template molecules provide reference information that feeds back into the quantitation calculation. This internal feedback mechanism allows the system to continuously correct for amplification bias based on the observed versus expected ratios of template molecules, improving reliability without requiring external data
3Productivity
If multiplex PCR is used to amplify multiple V and J segments, then the productivity of immune cell characterization is improved, but the amplification bias increases making accurate quantitation difficult
Solution Approach 1:
The patent applies segmentation by dividing the multiplex PCR amplification into individually trackable segments through unique barcode assignment. Each synthetic template molecule carries a unique barcode that identifies its specific V and J segment combination, allowing the system to monitor and correct amplification bias for each segment independently while maintaining the productivity benefits of multiplex amplification
Solution Approach 2:
The patent changes the parameter of trackability by incorporating unique barcode sequences into each synthetic template. This parameter change enables the system to distinguish between different V and J segment amplifications, allowing separate correction of amplification bias for each segment while maintaining high productivity through simultaneous multiplex amplification
4Measurement precision
If synthetic templates with unique barcodes are used for absolute quantitation, then the measurement precision is improved, but the device complexity increases due to need for template design and sequencing
Solution Approach 1:
The patent uses copying by creating synthetic template molecules that replicate the essential features of native immune cell DNA sequences but with added barcode identifiers. These copied templates maintain the same amplification characteristics as real templates while adding trackability through barcodes, enabling accurate quantitation without requiring complex analysis of native sequences. The copying approach simplifies the overall system by using standardized synthetic molecules rather than requiring complex native sequence analysis
Data Source
AI summary
Methods are provided for correction of amplification bias and quantitation of adaptive immune cells in a sample using synthetic templates that include random oligonucleotide sequences.