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• W2512011504 abstract "Aim We previously reported on the use of the Ion PGM next generation sequencing (NGS) platform to genotype HLA class I and class II genes by a super-high resolution, single-molecule, sequence-based typing (SS-SBT) method. However, HLA alleles could not be assigned at the field 4 level at some HLA loci such as DQA1, DPA1 and DPB1 because the SNP and indel densities were too low to identify and separate both of the phases. In this regard, we have now added the single molecule, real-time (SMRT®) DNA sequencer PacBio RS II method to our analysis in order to test whether it might determine the HLA allele sequences in some of the loci with which we previously had difficulties. Here, we report on sequence-based genotyping from the promoter-enhancer region to 3 ′ UTR of the major HLA genes in the Japanese using the PacBio RS II and Ion PGM NGS systems. Method Forty-six DNA samples were obtained from a reference set used previously to establish the HLA allele frequency data in the Japanese population. The reference samples represented more than 99.5% of the HLA alleles at each of the nine HLA loci. The genomic DNA samples were amplified by long ranged PCR using eleven HLA loci specific primer pairs (A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1 and DPB1). After NGS, consensus sequences were obtained via Long Amplicon analysis such as overlapping, clustering of filtered sub-reads, phasing and removing PCR artifacts. The HLA allele sequences for generating a library of consensus sequences were determined by mapping of sequence reads obtained from Ion PGM sequencer. Results A total of 219 HLA allele sequences (20 A, 40 B, 23 C, 31 DRB1, 36 DQA1, 14 DQB1, 15 DPA1 and 40 DPB1) that covered the promoter-enhancer region to 3’UTR were determined for the 46 samples. The classification of the newly identified SNPs and/or indels revealed at least three non-synonymous substitutions for one B and two DPA1 alleles, although most of the substitutions were observed in intron regions. Conclusion We determined at least 219 Japanese major HLA alleles at the field 4 level by NGS and we expect that this HLA genotyping method of entire HLA gene regions by NGS will help to precisely detect rare, novel and null alleles in population genetic and disease studies." @default.
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• W2512011504 date "2016-09-01" @default.
• W2512011504 modified "2023-09-27" @default.
• W2512011504 title "P019 Collection of major HLA allele sequences in japanese population towards precise NGS based HLA DNA typing at the field 4 level" @default.
• W2512011504 doi "https://doi.org/10.1016/j.humimm.2016.07.084" @default.
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