Multiplex KIR Genotyping via Emulsion PCR and Pyrosequencing
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Solution Overview
Problem
Current methods are inefficient in determining all KIR alleles present in patient samples in a time-efficient manner, limiting the ability to genotype KIR genes simultaneously across multiple individuals.
Innovation Solution
The method employs multiplex, parallel clonal sequencing analysis using pyrosequencing technology and emulsion PCR to amplify and sequence KIR genes, enabling the simultaneous typing of all 16 KIR genes across multiple individuals by generating long reads and resolving phase ambiguity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current methods are used to determine all KIR alleles in patient samples, then complete allele identification is achieved, but the process is time-consuming and inefficient
Solution Approach 1:
The patent segments the KIR gene family into 16 individual gene targets and uses multiple gene-specific primers to amplify each gene separately in a multiplex PCR reaction. This segmentation allows parallel processing of all KIR genes simultaneously, dramatically reducing the time required compared to sequential analysis while maintaining complete allele identification coverage
Solution Approach 2:
The patent combines multiple KIR gene amplification reactions into a single multiplex PCR reaction and pools the amplicons from multiple individuals into a single sequencing reaction. This merging approach enables simultaneous analysis of all 16 KIR genes across multiple samples in parallel, achieving high-throughput genotyping without sacrificing measurement precision
2Measurement precision
If KIR genes are sequenced individually, then accurate allele determination is achieved, but the throughput is limited and cannot genotype multiple individuals simultaneously
Solution Approach 1:
The patent pools KIR amplicons from multiple individuals into a single sequencing reaction, allowing simultaneous sequencing of all samples in parallel. Individual identification sequences in the primers enable bioinformatic separation of reads by individual, achieving high throughput while maintaining accurate genotype determination for each sample
Solution Approach 2:
The patent introduces individual identification sequences as intermediary tags in the primers. These tags act as mediators that allow mixed amplicons from multiple individuals to be accurately sorted and assigned to the correct individual during data analysis, enabling multiplexing without sacrificing genotype accuracy
3Speed
If short reads are used for sequencing, then the sequencing process is faster, but phase ambiguity cannot be resolved for KIR alleles
Solution Approach 1:
The patent performs emulsion PCR to generate long amplicons that span the entire KIR gene region before sequencing. This preliminary action of creating long templates ensures that phase information is preserved in the long reads, allowing unambiguous allele determination while maintaining efficient sequencing throughput
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 allows for high-throughput KIR genotyping of up to 10 individuals in a single sequencing run, providing unambiguous allele identification and improving the efficiency of disease association studies.
Implementation Method 1
determining the sequence of each KIR amplicon for each individual using pyrosequencing in parallel
Implementation Method 2
performing emulsion PCR
Data Source
AI summary
Disclosed is a method of determining KIR genotypes for one or more individuals in parallel, the method comprising: for each individual, amplifying the polymorphic exon sequences of the KIR genes, pooling the KIR amplicons, performing emulsion PCR followed by pyrosequencing in parallel to determine all the amplicon sequences present in the individual to determine which KIR alleles are present in the individual.