P166 Validation of a next generation sequencing based HLA typing system

Abstract

Aim Very high resolution typing of HLA genes by Next Generation Sequencing (NGS) methods is becoming mainstream in histocompatibility laboratories for the workup of haematopoietic stem cell and solid organ transplant patients and donors. Determining the full sequence for class I and extended exon coverage for class II genes produces HLA types with far fewer ambiguities than traditional Sanger sequencing based typing (SBT) and other lower resolution typing methods. In addition NGS brings benefits of higher throughput and lower cost. Methods We have validated the Illumina TruSight HLAv2 typing kit for HLA-A, B, C, DRB1/3/4/5, DQA1, DQB1, DPA1 and DPB1. A total of 151 samples were tested following the method described by the vendor. All method steps were carried out manually. Testing comprised 6 batches of 24 samples and 1 batch of 12 samples, which were made up of 73 local DNA samples typed previously by a combination of SBT, SSP and SSO, 54 DNA samples from the UCLA reference panel and 24 samples from the 17th International HLA Workshop Study blinded proficiency panel. Reference types for all eleven loci were not available for all local samples, four digit types were available for all eleven loci for the UCLA samples. Sequence analysis and genotype calling was performed using Illumina Assign for Trusight HLA v2.1 and GenDX NGSEngine. HLA type concordance rate, ambiguity rate and accuracy were examined. Results Total percent concordance between the reference type (n = 130) and genotype determined by Assign was as follows: A: 100; B: 99.2; C: 99.2; DRB1: 97.6.5; DRB345: 98.4; DQA1: 99.2; DQB1: 100; DPA1: 100; and DPB1: 97.6. No allele (1st, 2nd and 3rd field) or genotype ambiguity was found for HLA-A, B, C, DRB345 DQB1 or DPA1. An ambiguous type was determined in 4 cases for DRB1, 3 cases for DQA1 and 29 cases for DPB1. In the case of DRB1 and DPB1 this was due to lack of part of the gene sequence and for DQA1 it was due to poor read depth in those samples. Average read depth across all loci was 223. Repeat rates varied between 1.3% (A & B) and 10.9% (DPA1). Conclusions The Illumina TruSight HLAv2 typing kit provides a suitable first pass high resolution typing system for stem cell and solid organ transplant patients and donors. Accuracy is excellent and the ambiguity levels are very low. We intend to introduce this method for routine HLA typing.