P108 From the sample to donor/recipient compatibility: A comprehensive range of laboratory reagents and equipment for NGS-based genotyping of classical and non-classical HLA genes

Abstract

Aim The French organization for blood transfusion (EFS), developed a national research and development program to provide a complete range of reagents and instrumentation leading to a fast, simple and robust NGS strategy for HLA genotyping. Methods The specially-designed “NG-Mix PCR reagent” amplifies all classical Class I (HLA-A, B, C) and Class II (HLA-DRB1,DRB3, DRB5, DQA1, DQB1, DPA1, DPB1) genes as well as the non-classical HLA genes (HLA-E, F, G, DQA2, DQB2). Amplicon fragmentation is achieved with “NG-Frag”, a novel instrumentation recently developed by EFS which, in less than one minute, allows ultra-sonication based fragmentation of 96 samples loaded in a PCR plate in a unique single step. This highly standardized sonication step generates fragments in a uniform range of optimal size (400–800 bp), which eliminates the requirement to perform time-consuming fragment size selection and purification steps. The library is subsequently generated by using “NG-Lib”, which are specific reagents that produce a normalized library without the need to perform any extra PCR reactions for tagging in less than one hour for 96 samples. Library molarity is consistently and reliably quantified using “NG-Quant”, a q-PCR method using a specific probe (without presuming a hypothetical size range for library fragments, counter to other quantification technique). The sequencing reactions are performed on the MiSeq system (Illumina) before analysis with “NG-View”, a data compilation and interpretation software designed by the EFS. Results This method was used to generate results on 41 homozygous reference cell lines and a panel of 190 random BMVD samples previously genotyped by another NGS commercial product (Omixon). These results will be presented along with three other important aspects of the EFS NGS product: the technical advantages of the EFS NGS approach at each step of the process, a description of DQA2/DQB2 allelic system, and the observed linkage disequilibrium between classical and non-classical HLA genes. Furthermore, during the development of the EFS NGS product, numerous new classical and non-classical alleles were identified and submitted to the IMGT/HLA allele database. Conclusions At the time of preparation of this abstract, 52 alleles discovered by this method are already listed on the database.