P090 : NEXT-GENERATION SEQUENCING-BASED HLA TYPING OF SALIVA AND BLOOD SAMPLES FROM THE SAME DONORS PRODUCES CONCORDANT TYPING RESULTS

Abstract

Aim We present the results of next-generation sequencing (NGS) based HLA typing, using paired blood and saliva samples to demonstrate that typing results are independent of tissue source. We show that NGS reads from whole-genome sequencing (WGS) reads are also suitable for HLA typing, similarly to reads produced by well-targeted HLA kits albeit with lower precision. Methods Both saliva and blood samples were collected from two families. All samples were sequenced by both WGS and using a specific kit targeting the HLA-A, B, C and HLA-DRB1, -DQB1 genes. Reads obtained from the WGS kit were filtered for reads aligning to the IMGT/HLA database, and this subset was used as an input for the HLA typing algorithms. Two typing approaches were used, the first is based on read coverage statistics, the second method is building a consensus sequence that can be searched in the IMGT/HLA database. Results There was no significant difference in precision and accuracy between the results of blood and saliva samples. The choice of sequencing method had a much greater effect on precision. The different typing algorithms produced similar results, the most visible difference being the number of ambiguities reported. The two sequencing approaches gave nearly identical results, however the precision of typing obtained from WGS reads is lower due to a lower number of reads suitable for HLA typing. Nevertheless, four digit typing was possible in most cases. Targeting of HLA genes allowed 8 digit precision in some samples and ambiguity was lower for both typing algorithms. Targeted sequencing makes it possible to resolve phase ambiguities and detect novel alleles, however WGS was able to provide information about other genes present in IMGT/HLA (MICA, MICB TAP1, TAP2 etc.). Conclusion HLA typing results from blood and saliva samples are indistinguishable. The number and quality of reads obtained by WGS and targeted sequencing are significantly different. The quality control measures built-in the typing algorithm provide valuable threshold values for different statistics to reduce ambiguities. WGS with 30–40× average coverage is not suitable for high precision HLA typing – a targeted kit should be used to get 6 or 8 digit results – but the WGS approach can gather information about wider range of genes in the IMGT/HLA database.