HLA Gene Amplification Primers for Multiplex Sequencing
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Solution Overview
Problem
Current HLA genotyping methods face challenges such as low accuracy, inability to detect new alleles, and ambiguous results due to limitations in sequencing technologies like SSO, SSP, SBT, and NGS, particularly in phasing and resolving heterozygote samples, and require multiple tubes for amplification, making the process complex and inefficient.
Innovation Solution
The development of HLA gene amplifying primers and a sequencing library construction method using PacBio's third-generation single-molecule real-time sequencing technology, which includes nine primer sets for multiplex PCR amplification, enabling simultaneous amplification of 11 HLA gene sequences in a single tube and providing comprehensive information about both exon and intron regions, improving accuracy and simplifying the process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If SSO or SSP methods are used for HLA genotyping, then the experimental process is simple and the cycle is short, but the ability to detect new alleles is lost and cross-reaction occurs
Solution Approach 1:
The patent combines the advantages of SSO/SSP (simplicity, speed) with SBT (new allele detection) by using a unified NGS-based platform that incorporates optimized primers and library construction methods, achieving both rapid processing and high accuracy in detecting known and new alleles
Solution Approach 2:
The patent optimizes key parameters including primer sequences and lengths, PCR cycling conditions, and library construction protocols to enable efficient amplification and sequencing that maintains speed while improving detection accuracy and new allele identification
2Reliability
If SBT method is used, then new alleles can be detected, but phasing cannot be performed and heterozygote samples produce ambiguous results
Solution Approach 1:
The patent transitions from traditional SBT's single-dimensional sequencing approach to a multi-dimensional solution by combining long-read sequencing capabilities with optimized primer designs that capture phased haplotype information, enabling resolution of heterozygote samples through extended read lengths that span multiple polymorphic sites
Solution Approach 2:
The patent introduces specialized adapter sequences and optimized library construction protocols as intermediaries that facilitate proper fragment preparation and sequencing, enabling accurate phasing and resolution of ambiguous heterozygote results while maintaining new allele detection capability
3Reliability
If NGS technology is used, then new alleles can be detected, but ambiguous results occur due to short read length and data calculation dependency
Solution Approach 1:
The patent fundamentally changes the read length parameter by utilizing long-read sequencing technology (e.g., PacBio, Oxford Nanopore) that generates reads spanning several kilobases, eliminating the need for complex assembly algorithms and directly providing phased haplotype information with high typing resolution
Solution Approach 2:
The patent replaces the computational assembly process (data calculation dependency) with a direct physical sequencing approach where long reads naturally capture complete haplotype information, reducing reliance on complex bioinformatics assembly algorithms and minimizing ambiguous results
4Reliability
If multiple tubes are used for amplification of different HLA genes, then comprehensive coverage is achieved, but the process becomes complex and inefficient
Solution Approach 1:
The patent merges multiple separate amplification reactions into a single multiplex PCR reaction by designing a set of primers that simultaneously target all 11 HLA genes (HLA-A, B, C, DPB1, DQA1, DQB1, DPA1, DRB1, DRB3, DRB4, DRB5), reducing the number of tubes and steps while maintaining comprehensive gene coverage
Solution Approach 2:
The patent creates a universal primer set that can amplify all HLA genes of interest in a single reaction, making the protocol universally applicable to comprehensive HLA typing without requiring gene-specific optimization of multiple separate reactions, thereby simplifying the overall process
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 enhances the accuracy of HLA genotyping by achieving 3-field resolution without ambiguous results, simplifies the experimental process, and provides complete HLA gene information, improving sensitivity and typing accuracy through long read lengths and multiplex amplification capabilities.
Implementation Method 1
HLA gene amplifying primers, including primers in any one or more of the following nine primer sets... subjected a test sample to amplification using the HLA gene amplifying primers to obtain an amplification product
Implementation Method 2
using PacBio's third-generation single-molecule real-time sequencing technology... providing complete HLA gene information
Data Source
AI summary
Provided is an HLA gene amplification primer, comprising primers of any one or more groups of the following nine primer groups: a first primer group: SEQ ID NO: 1 and SEQ ID NO: 2; a second primer group: SEQ ID NO: 3 and SEQ ID NO: 4; a third primer group: SEQ ID NO: 5 and SEQ ID NO: 6; a fourth primer group: SEQ ID NO: 7 and SEQ ID NO: 8; a fifth primer group: SEQ ID NO: 9 and SEQ ID NO: 10; a sixth primer group: SEQ ID NO: 11 and SEQ ID NO: 12; a seventh primer group: SEQ ID NO: 12 and SEQ ID NO: 13; an eighth primer group: SEQ ID NO: 14 and SEQ ID NO: 15; and a ninth primer group: SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18. Also provided is a kit for HLA gene sequencing, which comprises the described HLA gene amplification primer; provided is a gene sequencing library construction method based on the described HLA gene amplification primer and an HLA gene sequencing method based on the HLA gene sequencing library construction method.