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Updated: Dec 14, 2025

Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing
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Genomic characterization of MICA gene using multiple next generation sequencing platforms: A validation study.

Yizhou Zou1, Jamie L Duke2, Deborah Ferriola2

  • 1Department of Immunology, Central South University Xiangya School of Medicine, Changsha, Hunan, China.

HLA
|July 19, 2020
PubMed
Summary

We developed a next-generation sequencing (NGS) protocol for comprehensive MICA gene genomic characterization. This method accurately sequences the full MICA gene, improving allele resolution and database completeness.

Keywords:
IlluminaMICANGSOxford Nanoporegenotyping

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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.