P100 Complete single mica gene sequencing-based typing using next generation sequecers

Abstract

Aim The MICA gene located in the HLA gene cluster region close to HLA-B is highly polymorphic. It is associated with solid organ transplant rejection and bone marrow transplant GVHD. To test a novel Next Generation Sequencing (NGS) based MICA high resolution typing method, we used both Sanger Based Typing (SBT) and NGS in a parallel double blind study. Methods Genomic DNA samples from 54 healthy Han people in southern China were genotyped by both SBT and NGS methods. Well established MICA SBT was performed using an amplicon covering the MICA gene from exon 2 to exon 5 (2.2kb) and MICA-Seq specific software. NGS was performed with a PCR product that includes the entire length of the MICA gene from mid 5’UTR to the end of the 3’UTR (12.6 Kb). Library preparation was performed with the standard library preparation method and reagents from Omixon’s V2 Holotype HLA kits and sequenced on an Illumina MiSeq using paired-end 2x250 bp V2 chemistry. MICA typing was assigned using Omixon Twin software and GenDx’s NGSEngine. Results 2.04 million reads with an average quality score ⩾Q30 were generated. 93.3% of reads produced were high quality and used for MICA consensus assembly with an average coverage depth of 300 across the exonic regions. 26 MICA genotypes consisting of 17 unique alleles among 54 samples were identified, and were 100% concordant between NGS and SBT. MICA NGS was able to resolve all ambiguities obtained by MICA SBT. Alleles initially typed as ambiguous MICA*008:01/04 by SBT were determined to be MICA*008:01:01, *008:01:02 and *008:04. MICA*008:04 was the most frequent at 19.4% (21/108 alleles) by NGS. In addition, 4 samples typed ambiguously as MICA*002:01/MICA*008 or MICA*023/MICA*035, and 3 samples typed as MICA*002:01/MICA*010 or MICA*022/MICA*025 by SBT were resolved by NGS. Furthermore a new *008:01 allele was found with 5 differences in intron 1 and 1 difference in intron 5. This new allele was represented three times in the population of 108 allele calls. Additionally we were able the complete the sequencing of exon 1 for *009:01 and *033 and exon 6 for *007:01 and *033 alleles. Conclusions A NGS based MICA typing method has been established and is capable of resolving ambiguities observed with SBT, identifies new alleles and allows the completion of IMGT reported incomplete allele sequences.