Concordance of next generation sequence-based and sequence specific oligonucleotide probe-based HLA-DRB1 genotyping

Area of Science:

• Immunogenetics
• Genomics
• Molecular Biology

Background:

• Human Leukocyte Antigen (HLA) genotyping is crucial for transplantation and disease association studies.
• Previous HLA genotyping often relied on moderate-resolution methods like sequence-specific oligonucleotide probe (SSOP) technology.
• Next-generation sequencing (NGS) offers higher resolution but requires validation against established methods.

Purpose of the Study:

• To compare the accuracy and concordance of HLA genotyping between NGS and SSOP methods.
• To assess the impact of NGS's higher resolution on existing genotype data.
• To identify sources of error in HLA genotyping and propose improvements.

Main Methods:

• Genotyping of HLA-DRB1, DRB3, DRB4, and DRB5 loci (exon 2) using NGS (Roche 454) on 993 African American newborn samples.
• Comparison of NGS genotype calls with previously generated SSOP data for the same samples.
• Analysis of discordant genotypes to determine the cause of discrepancies.

Main Results:

• A concordance rate of 92.3% was observed between NGS and SSOP genotyping.
• Only 3 out of 76 discordant genotypes were due to the higher resolution of NGS.
• The majority of discordances (73/76) resulted from preventable errors in sample handling, data interpretation, or entry.
• NGS-based methodology yielded data of superior quality and accuracy.

Conclusions:

• NGS provides more accurate HLA genotyping data compared to SSOP.
• Errors in sample handling and data management significantly impact genotyping accuracy.
• Improving genomic DNA quality, automating processes, and utilizing high-resolution technology are key to enhancing HLA data quality.