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

Area of Science:

• Immunogenetics
• Genomics

Background:

• The Major Histocompatibility Complex Class I chain-related gene A (MICA) plays a role in immune responses.
• Accurate MICA gene characterization is crucial for understanding its role in various diseases and transplantation outcomes.
• Existing methods for MICA genotyping have limitations in resolving complex polymorphisms and full-length gene sequences.

Purpose of the Study:

• To establish and validate a robust next-generation sequencing (NGS) protocol for the complete genomic characterization of the MICA gene.
• To enhance the accuracy and comprehensiveness of MICA allele sequencing compared to legacy methods.
• To contribute novel MICA sequences and allele information to the IMGT/HLA database.

Main Methods:

• Development of a 13kb amplicon encompassing the full MICA gene.
• Application of a dual-NGS approach using Illumina MiSeq and Oxford Nanopore Technology (ONT) MinION platforms.
• Validation of the NGS assay using 97 samples previously characterized by Sanger or SSO methods.
• Genotyping of 59 additional diverse DNA samples of unknown ethnicity.

Main Results:

• The NGS protocol demonstrated high accuracy, precision, specificity, and sensitivity in MICA gene characterization.
• Complete consensus sequences from 5' UTR to 3' UTR were generated for all sequenced alleles using ONT.
• Thirty-two novel or gap-filled MICA sequences were submitted to the IMGT/HLA database, including new alleles and extended sequence information.
• Successful phasing of multiple polymorphisms and resolution of ambiguities were achieved.

Conclusions:

• The developed NGS protocol provides a powerful and accurate method for comprehensive MICA gene sequencing.
• This approach significantly improves the characterization of MICA alleles, contributing valuable data to the IMGT/HLA database.
• The study highlights the utility of combining different NGS technologies for resolving complex genetic variations and generating complete gene sequences.