P109 Validation of genotyping method for human leukocyte antigen based on next generation sequencing technology

Abstract

Next generation sequencing (NGS) technology allows genotyping for Human Leukocyte Antigen (HLA) genes with full resolution for multiple samples in a single run. Compared to traditional methods of SSO, SSP and Sanger sequencing, NGS delivers more complete coverage of the genome and phased contig sequences to resolve ambiguities. Despite the advantages of the NGS technology, validation at 3 and 4 field resolution of the HLA typing results is challenging because other technologies provide only up to 3 field resolution. We first introduce a HLA typing method based on NGS developed for high throughput applications and then the accuracy is assessed through pedigree analysis on a large cohort of family trios from a disease association study. This high throughput typing method can generate accurate genotype calls for all 11 major HLA genes with 4 field resolution based on NGS technology. For data analysis, three orthogonal algorithms are combined to rank the genotype candidates and generate consensus sequences for individual alleles. Ambiguity is resolved for heterozygous sample by phasing analysis except for certain allele combinations in DPB1. The method can be used to type 96–384 samples in a single sequencing run for high throughput HLA typing applications, including registry typing, disease association and population studies. For family trios, the accuracy of genotyping results at 3 and 4 field resolution is assessed through pedigree analysis. Approximately 1000 family trios are typed by the above method. At 3 field resolution, the validated genotyping accuracies for automatic calls are above 98.8% and 98.3% for class I and class II genes respectively. For reviewed calls, the validated accuracies are around 99.9% and 99.8%. The concordance between automatic and reviewed calls is above 99.4%. The validation based on pedigree analysis provides a way to assess the accuracy of HLA genotyping methods with 3 and 4 field resolution. The high concordance rate between automatic and reviewed calls showed that the software provides high quality calls and the review process is made effortless for trained technicians. H. Ollila : Grant/Research Support; Company/Organization; 5R01MH096262 . D. Levinson : Grant/Research Support; Company/Organization; 5R01MH096262.