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• Published in: Human immunology Vol. 74; p. 38
• Main Authors: Duke, Jamie L, Sasson, Ariella, Mackiewicz, Kate, Lind, Curt, Major, Endre, Hague, Tim, Berces, Attila, Monos, Dimitri
• Format: Journal Article
• Language: English
• Published: 01.11.2013
• Subjects: Allergy and Immunology
• ISSN: 0198-8859
• DOI: 10.1016/j.humimm.2013.08.059

Aim Capture and sequence by NGS the 4Mb of the MHC and derive genotypes of 33 MHC genes (HLA and non-HLAs) using Omixon Target HLA Typing software. Methods We applied our Region Specific Extraction method to target and capture 4Mb of the MHC region with custom designed capture oligos on 7 samples having known HLA genotypes: 5 homozygous cell lines (COX, DBB, JVM, PGF, and QBL) and 2 heterozygous samples (mixture of PGF and COX DNA, and a standard lab control). Each sample was whole-genome amplified, prepared for sequencing using Illumina TruSeq DNA sample prep kit and size selected for DNA fragments ranging from 300-400 bp. Paired-end 2x100bp sequencing was performed on the Illumina HiSeq. Each sample was genotyped for 33 genes within the MHC by Omixon Target using all paired-end reads. Derived genotypes were compared to known genotypes from the IMGT/HLA database for the cell lines and Sanger sequencing for the heterozygous control sample. Results We sequenced 7 samples throughout the 4Mb of the MHC region. On average, each sample had 284 million reads which were analyzed with Omixon Target. The 33 genes examined were Class I HLA-A, B, C, E, F, G, H, J, K, L, and V, Class II genes HLA-DMA, DMB, DOA, DOB, DPA1, DPB1, DQA1, DQB1, DRA, DRB1, DRB3, DRB4, DRB5, Class II pseudogenes HLA-DRB2, DRB6, DRB7, DRB8, DRB9, and MHC related genes MICA, MICB, TAP1, and TAP2. Omixon Target provided genotypes for 90% of the possible alleles; the remaining 10% of alleles (no genotype) were Class II pseudogenes. The average depth across the alleles ranged from 50–3000, a small percentage of the overall reads per sample. In the cases where the true HLA genotype was known, we determined the correct allele with 93% accuracy. Conclusions Our method of capturing and sequencing the MHC coupled with Omixon Target has yielded highly accurate genotypes for 33 HLA and non-HLA, MHC genes. This work sets the stage for further detailed characterization of the MHC and derivation of clinically relevant HLA typing from MHC NGS data. Major: Omixon, Inc.: Other: Contractor. Hague: Omixon, Inc.: Other: Contractor. Berces: Omixon, Inc.: Employee; Stockholder.