HLA-genotyping of clinical specimens using Ion Torrent-based NGS

Area of Science:

• Immunogenetics
• Molecular Diagnostics
• Genomics

Background:

• Accurate human leukocyte antigen (HLA) typing is crucial for transplantation and disease association studies.
• Next-Generation Sequencing (NGS) offers a high-resolution approach for HLA genotyping.
• The NXType™ workflow and TypeStream™ software provide an integrated NGS solution for HLA analysis.

Purpose of the Study:

• To evaluate and validate the performance of the NXType™ workflow and TypeStream™ software on the Ion Torrent NGS platform.
• To assess the accuracy and reliability of the system across diverse human populations and challenging genetic scenarios.
• To determine the turnaround time and identify potential limitations of the NGS-based HLA typing system.

Main Methods:

• Utilized a comprehensive validation panel of 285 genomic DNA (gDNA) samples from diverse ethnic populations.
• Included 59 proficiency testing (PT) samples and 226 clinical specimens, covering 3420 alleles across six HLA loci.
• Compared NGS-derived genotypes with those obtained from sequence-based typing (SBT) as a reference method.

Main Results:

• Achieved high concordance rates between NGS and SBT: 99.5%–99.8% for HLA-A, B, C, DRB1, DQB1, and 98.9% for HLA-DPB1.
• The NXType™ system accurately genotyped a wide spectrum of HLA variations, including rare and novel alleles.
• The complete NGS workflow, from amplification to genotype assignment, was completed within 3 working days.

Conclusions:

• The NXType™ workflow and TypeStream™ software demonstrate robust performance for high-resolution HLA typing on the Ion Torrent NGS platform.
• The system is capable of accurately identifying diverse HLA alleles, supporting its use in clinical and research settings.
• Identified areas for further improvement in the platform, assay, and software algorithms based on validation findings.