Multiplexed PCR Amplification Bias Correction via Template Standards
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for high-throughput sequencing of adaptive immune receptor encoding DNA face challenges in accurately quantifying the diversity of TCR and BCR sequences due to biased primer utilization in multiplexed amplification reactions, leading to skewed representation of individual sequences.
Innovation Solution
A composition and method that standardize the amplification efficiency of oligonucleotide primers using a template oligonucleotide set with diverse sequences, ensuring equimolar representation and barcode sequences for precise identification and quantification of V and J region sequences, thereby correcting for non-uniform amplification potential.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiplexed amplification reactions are used to amplify diverse immune receptor sequences, then the throughput and efficiency of sequencing is improved, but amplification bias occurs leading to non-uniform representation of different sequences
Solution Approach 1:
The patent applies preliminary action by performing amplification bias correction through computational algorithms before final quantification of sequence diversity. The method calculates expected amplification efficiencies based on known primer-template interactions and uses these predictions to correct the observed sequencing data, thereby removing the distortion caused by multiplexed amplification bias while preserving the high throughput benefit
Solution Approach 2:
The patent implements feedback by using the sequencing results themselves to identify and correct amplification biases. The method compares observed sequence frequencies with expected frequencies based on input template composition, detects deviations caused by primer bias, and applies corrective factors to recover accurate quantification of the original immune receptor diversity
2Device complexity
If standard multiplexed PCR is used for amplification, then the complexity of the procedure is reduced, but the quantification of immune receptor sequences becomes skewed due to biased primer utilization
Solution Approach 1:
The patent introduces an intermediary computational correction step that mediates between the simple multiplexed PCR amplification and the final quantification. This intermediary process uses bioinformatics algorithms to calculate and apply bias correction factors, allowing the use of simple multiplexed PCR while achieving accurate quantification through computational compensation for the amplification biases introduced
3Adaptability or versatility
If diverse oligonucleotide primers are used to amplify all V and J regions, then the coverage of immune receptor diversity is improved, but the uniformity of amplification across different primers deteriorates
Solution Approach 1:
The patent applies parameter changes by systematically varying primer concentrations and lengths across the diverse primer set to optimize amplification uniformity. The method adjusts these parameters based on empirical performance data and computational predictions, allowing maintenance of comprehensive V and J region coverage while improving the stability and uniformity of amplification across all primers through optimized design parameters
Data Source
AI summary
Compositions and methods are described for standardizing the DNA amplification efficiencies of a highly heterogeneous set of oligonucleotide primers as may typically be used to amplify a heterogeneous set of DNA templates that contains rearranged lymphoid cell DNA encoding T cell receptors (TCR) or immunoglobulins (IG). The presently disclosed embodiments are useful to overcome undesirable bias in the utilization of a subset of amplification primers, which leads to imprecision in multiplexed high throughput sequencing of amplification products to quantify unique TCR or Ig encoding genomes in a sample. Provided is a template composition comprising a diverse plurality of template oligonucleotides in substantially equimolar amounts, for use as a calibration standard for amplification primer sets. Also provided are methods for identifying and correcting biased primer efficiency during amplification.