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

Abstract

Next generation sequencing (NGS) of clonally amplified DNA, using Roche 454 technology, was used to genotype HLA-DRB1, DRB3, DRB4, and DRB5 loci (exon 2 only) from a set of 993 samples from newborns with maternally-reported African American ancestry. DRB1 exon 2 was genotyped previously on the same sample set using sequence-specific oligonucleotide probe (SSOP) technology. Comparison of the genotype calls from both methods indicated concordance of 92.3%. Some discordance was expected due to the higher resolution of NGS data, compared to SSOP data. This resulted from selection of the incorrect allele from the ambiguity string produced by SSOP genotyping. Of 76 discordant genotypes, only three were due to resolution of ambiguity with the NGS method. The low percent of changes due to the increased resolution of the NGS method instills confidence in the overall value of previous data genotyped with moderate resolution methods, i.e., the vast majority of alleles present in a population are those that are detectable at moderate resolution. The remaining 73 discordant genotypes resulted from preventable errors in sample handling, data interpretation, and data entry. These results underscore the potential for error that can result from factors such as low quality genomic DNA, manual data entry, and interpretation of marginal genotyping results. Optimization of genomic DNA quality, automation of genotyping steps wherever possible, and use of the highest resolution technology available can lead to dramatic improvements in HLA genotype data quality. NGS-based methodology generated data of superior quality and accuracy compared to the SSOP system.

Performance Characteristics and Validation of Next-Generation Sequencing for Human Leucocyte Antigen Typing

“…A thorough validation should not only determine whether an assay performs robustly, but also identify areas of concern. Consistent with previous reports, HLA typing of DRB1/3/4/5 was the most difficult 22,24,25 (Table 2). These issues arise because of primer locations inhibiting the ability to resolve certain HLA alleles.…”

“…TruSight HLA demonstrated efficient generation of long-range HLA amplicons (Figure 1) and NGS libraries that consistently generated high-quality data (Figures 2 and 3). The degree of concordance is consistent with previous reports comparing different sequencing platforms and NGS HLA assays 17,21,22 (Table 2). The costeffectiveness of HLA typing by NGS is consistent with a report by Stoddard et al 23 on the use of NGS compared to Sanger for diseases with multiple candidate genes (Figure 4).…”

Evaluation of computational programs to predict HLA genotypes from genomic sequencing data

“…This is somewhat surprising as Lane et al [ 33 ] compare the HLA status derived from SSOP against clonally amplified DNA, using Roche 454 technology for 993 samples from newborns with maternally reported African American ancestry and found a concordance of 92.3%. However, it is consistent with Gourley et al [ 34 ], who reported typing discrepancies between 3.9% and 6.7% for HLA-A and B, respectively, based on SSOP typing of 1983 samples.…”

Genetic fine mapping of systemic lupus erythematosus MHC associations in Europeans and African Americans

“…We generated a new reference panel of HLA-typed individuals in a subset of the AA data. A total of 308 subjects were genotyped for classical class II HLA alleles (HLA * DQA, HLA * DQB and HLA * DRB1) by targeted sequencing of exons 2 and 3 ( HLA-DQA and HLA-DQB ) and exon 2 ( HLA-DRB1 ) ( 17 ). These were added to the database of reference HLA genotypes for HLA imputation with the software HLA * IMPV2 ( 18 ).…”

“…A total of 308 subjects were also genotyped for classical class II HLA alleles (HLA * DQA, HLA * DQB and HLA * DRB1) by targeted sequencing of exons 2 and 3 ( HLA-DQA and HLA-DQB ) and exon 2 ( HLA-DRB1 ) ( 17 ). This set included the ‘HLA reference set’ used for HLA imputation into the rest of the AA study.…”