NDLI: 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

Content Provider	Springer Nature : BioMed Central
Author	Ozaki, Yuki Suzuki, Shingo Kashiwase, Koichi Shigenari, Atsuko Okudaira, Yuko Ito, Sayaka Masuya, Anri Azuma, Fumihiro Yabe, Toshio Morishima, Satoko Mitsunaga, Shigeki Satake, Masahiro Ota, Masao Morishima, Yasuo Kulski, Jerzy K Saito, Katsuyuki Inoko, Hidetoshi Shiina, Takashi
Abstract	Background HLA 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. Results We 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. Conclusions In 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.
Related Links	https://bmcgenomics.biomedcentral.com/counter/pdf/10.1186/s12864-015-1514-4.pdf
Ending Page	12
Page Count	12
Starting Page	1
File Format	HTM / HTML
ISSN	14712164
DOI	10.1186/s12864-015-1514-4
Journal	BMC Genomics
Issue Number	1
Volume Number	16
Language	English
Publisher	BioMed Central
Publisher Date	2015-04-18
Access Restriction	Open
Subject Keyword	Life Sciences Microarrays Proteomics Animal Genetics and Genomics Microbial Genetics and Genomics Plant Genetics and Genomics Human Leukocyte Antigen Next Generation Sequencing Human Leukocyte Antigen Allele Phase Ambiguity Human Leukocyte Antigen Locus
Content Type	Text
Resource Type	Article
Subject	Biotechnology Genetics
Journal Impact Factor	3.5/2023
5-Year Journal Impact Factor	4.1/2023

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