OR15 New NGS HLA typing by targeted enrichment procedure, capture method

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Introduction NGS (next generation sequencing)-based HLA typing generally uses PCR reaction for enrichment of HLA genome fragments. However, PCR has some limitations which possibly cause HLA typing errors, such as chimeric sequences, mis-incorporation of nucleotides, allele drops, difficulties in optimization of PCR reaction and in automatization of HLA typing as routine works. In order to replace PCR step, the bead-based capture method for enrichment of HLA genome fragments was incorporated and developed as reliable and accurate HLA typing. Methods A total of 37 HLA genes including classical, non-classical and pseudo HLA genes (HLA-A, -B, -C, -E, -F, -G, MICA, MICB, HLA-H, -J, -K, -L, -V, -DRA, -DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1, -DPA1, -DPB1, -DMA, -DMB, -DOA, -DOB, -DQA2, -DQB2, -DRB2, -DRB6, -DRB7, -DRB8, -DRB9, -DPA2, -DPA3, -DPB2 and -DQB3) were selected and designed for capture probes with 120 bp in length. Genomic DNAs captured by hybridization with capture probes from 20 ng whole genome DNA were subjected to NGS sequencing for genomic sequence of the entire region of 37 HLA loci including the segments from the promoter-enhancer region flanking at the 5 ′ end of the gene to the flanking region at the 3 ′ end. For example, the 8 kb segment around the HLA-A gene with 5.5 kb in length was thus determined for genomic sequence. Results and conclusions Four field level typing (formerly, 8 digit) was established for allele typing of 37 HLA genes with concordance rate of 99.7%. Advantages of this capture method are (1) enabling genotyping as many as 37 HLA genes of 96 samples per one run, (2) no need of PCR step for HLA gene amplification which possibly generates chimeric sequences, mis-incorporation of nucleotides and allele drops, (3) easy operation and handling of only a single tube per one sample from the beginning, (4) easy to increase targeted genes, just by the addition of probes, (5) cost-effective, especially in the case of typing many samples, 2.0 US $/locus (96 samples/one run), (6) easy to automatize the full steps for the development of hands-free HLA typing using BIOMEK NXP and the KAPA HyperPlus kit. Recently, we have developed simultaneous 33 HLA genes and 18 KIR genes typing in a single tube per one run using this capture method just by the addition of KIR gene probes.