ArticleBMC genomics2015
Cost-efficient multiplex PCR for routine genotyping of up to nine classical HLA loci in a single analytical run of multiple samples by next generation sequencing.
Article in BMC genomics, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 18 papers.
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Who cites it
18 citing papers in PubMed.
- Optimized multiplex PCR-NGS for comprehensive HLA genotyping in Chinese populations: resolving ambiguities at high resolution.Frontiers in immunology · 2025Article
- Article
- Advancements in HLA Typing Techniques and Their Impact on Transplantation Medicine.Medical principles and practice : international journal of the Kuwait University, Health Science Centre · 2024Review
- Article
- Super high-resolution single-molecule sequence-based typing of HLA class I alleles in HIV-1 infected individuals in Ghana.PloS one · 2022Article
- Application of next-generation sequencing for the high-resolution typing of MHC-Frontiers in genetics · 2022Article
- Article
- Improving cord blood typing with next-generation sequencing: impact of allele-level HLA and NIMA determination on their selection for transplantation.Bone marrow transplantation · 2020Article
- Capturing Differential Allele-Level Expression and Genotypes of All Classical HLA Loci and Haplotypes by a New Capture RNA-Seq Method.Frontiers in immunology · 2020Article
- Identification of Novel Alleles and Structural Haplotypes of Major Histocompatibility Complex Class I and DRB Genes in Domestic Cat (Frontiers in genetics · 2020Article
- Long-read sequencing in deciphering human genetics to a greater depth.Human genetics · 2019Review
- Revisiting the potential power of human leukocyte antigen (HLA) genes on relationship testing by massively parallel sequencing-based HLA typing in an extended family.Journal of human genetics · 2019Article
- Evolutionary basis of HLA-DPB1 alleles affects acute GVHD in unrelated donor stem cell transplantation.Blood · 2018Article
- Use of coagulation factor XIII (F13) gene as an internal control for normalization of genomic DNA's for HLA typing.MethodsX · 2018Article
- Reference Grade Characterization of Polymorphisms in Full-Length HLA Class I and II Genes With Short-Read Sequencing on the ION PGM System and Long-Reads Generated by Single Molecule, Real-Time Sequencing on the PacBio Platform.Frontiers in immunology · 2018Article
- HLA genotyping by next-generation sequencing of complementary DNA.BMC genomics · 2017Article
- Article
- The impact of next-generation sequencing technologies on HLA research.Journal of human genetics · 2015Review
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18 authors.
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No grant is acknowledged in the PubMed record.
Abstract
backgroundHLA genotyping by next generation sequencing (NGS) requires three basic steps, PCR, NGS, and allele assignment. Compared to the conventional methods, such as PCR-sequence specific oligonucleotide primers (SSOP) and -sequence based typing (SBT), PCR-NGS is extremely labor intensive and time consuming. In order to simplify and accelerate the NGS-based HLA genotyping method for multiple DNA samples, we developed and evaluated four multiplex PCR methods for genotyping up to nine classical HLA loci including HLA-A, HLA-B, HLA-C, HLA-DRB1/3/4/5, HLA-DQB1, and HLA-DPB1.
resultsWe developed multiplex PCR methods using newly and previously designed middle ranged PCR primer sets for genotyping different combinations of HLA loci, (1) HLA-DRB1/3/4/5, (2) HLA-DQB1 (3.8 kb to 5.3 kb), (3) HLA-A, HLA-B, HLA-C, and (4) HLA-DPB1 (4.6 kb to 7.2 kb). The primer sets were designed to genotype polymorphic exons to the field 3 level or 6-digit typing. When we evaluated the PCR method for genotyping all nine HLA loci (9LOCI) using 46 Japanese reference subjects who represented a distribution of more than 99.5% of the HLA alleles at each of the nine HLA loci, all of the 276 alleles genotyped, except for HLA-DRB3/4/5 alleles, were consistent with known alleles assigned by the conventional methods together with relevant locus balance and no excessive allelic imbalance. One multiplex PCR method (9LOCI) was able to provide precise genotyping data even when only 1 ng of genomic DNA was used for the PCR as a sample template.
conclusionsIn this study, we have demonstrated that the multiplex PCR approach for NGS-based HLA genotyping could serve as an alternative routine HLA genotyping method, possibly replacing the conventional methods by providing an accelerated yet robust amplification step. The method also could provide significant merits for clinical applications with its ability to amplify lower quantity of samples and the cost-saving factors.
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