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

Abstract

We have developed a protocol regarding the genomic characterization of the MICA gene by next generation sequencing (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 sequence specific oligonucleotide) 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 (ONT), 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 ONT, extending from the 5′ untranslated region (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.

MICA and MICB allele assortment in Finland

“…Confirmatory sequencing of the novel alleles was performed at the Anthony Nolan Research Institute (London, UK). Full-length (5 0 -3 0 UTR) amplicons were generated by PCR amplification using in-house primers for MICB, and previously published primers for MICA 17 using VeriFi polymerase (PCR Biosystems, UK). In-house primer sequences can be made available upon reasonable request.…”

“…The data on MICA and MICB allele distribution in different populations are relatively limited compared to the classical MHC‐I alleles. The allele and haplotype frequencies reported vary notably between populations in Europe and Asia 15–30 . The Finnish population is a genetic mixture of Western and Eastern European populations with Siberian influence 31–34 with bottlenecks and sub‐isolates in history 35,36 .…”

“…The allele and haplotype frequencies reported vary notably between populations in Europe and Asia. [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] The Finnish population is a genetic mixture of Western and Eastern European populations with Siberian influence [31][32][33][34] with bottlenecks and sub-isolates in history. 35,36 Finland is also a genetic outlier in its HLA variation, with several HLA alleles or haplotypes enriched in Finland but rare elsewhere.…”

Development of a high-resolution mass-spectrometry-based method and software for human leukocyte antigen typing

“…The HLA locus is one of the most diverse and polymorphic regions in the human genome; more than 22,000 HLA alleles have been identified and reported by the IPD-IMGT/HLA database (IPD-IMGT/HLA release 3.47; Jan 2022) (10). To prevent alloreactivity, it is of crucial importance to reliably match the exact HLA allele type (11). For HLA typing, each HLA is assigned a unique name, which consist of four fields (an 8-digit number) of resolution separated by colons (i.e., HLA-A*01:01:01:01) (12).…”