Genomic characterization of MICA gene using multiple next generation sequencing platforms: A validation study.

We have developed a protocol regarding the genomic characterization of the MICA gene by NGS. The amplicon includes the full length of the gene and is about 13 kb. A total of 156 samples were included in the study. Ninety-seven of these samples were previously characterized at MICA by legacy methods (Sanger or SSO) and were used to evaluate the accuracy, precision, specificity, and sensitivity of the assay. An additional 59 DNA samples of unknown ethnicity volunteers from the United States were only genotyped by NGS. Samples were chosen to contain a diverse set of alleles. Our NGS approach included a first round of sequencing on the Illumina MiSeq platform and a second round of sequencing on the MinION platform by Oxford Nanopore Technology, on selected samples for the purpose of either characterizing new alleles or setting phase among multiple polymorphisms to resolve ambiguities or generate complete sequence for alleles that were only partially reported in the IMGT/HLA database. Complete consensus sequences were generated for every allele sequenced with Oxford Nanopore Technology, extending from the 5' UTR to the 3' UTR of the MICA gene. Thirty two MICA sequences were submitted to the IMGT/HLA database including either new alleles or filling up the gaps (exonic, intronic and/or UTRs) of already reported alleles. Some of the challenges associated with the characterization of these samples are discussed. This article is protected by copyright. All rights reserved.