P226 Investigation of loss of alleles in next generation sequencing HLA typing

Abstract

The HLA typing laboratory of the Hellenic Cord Blood Bank, has been using NGS technology for the routine typing of the Cord Blood Units and the paired mother samples since 2015. In November 2016, a run of 64 samples was performed with the Holotype 7-loci kit by Omixon. It was observed that the overall yield of the amplifications for HLA-A,-C,-DRB1,-DQA1, and DQB1 was not as uniform as it usually is, and there were a number of failed reactions for HLA-B and HLA DPB1. The failed reactions were repeated with acceptable results for HLA-B but 16 samples failed to amplify for a second time for HLA-DPB1. We proceeded with the library preparation for all samples and after the Miseq run, the analysis with HLA Twin v1.1.4.2. No results were obtained for 16 DPB1 loci as expected, as well as 1 B locus that had amplified very weakly. For 64 analyses we obtained 337 QC Passed loci, 81 loci with Warnings and 13 (7 DPB1, 4 B, 1 DRB1) Failed loci, according to the software “traffic light” system. While cross-checking the paired CBU/Mother typings, it was noted that for 7 pairs the results were discordant, with no shared allele in one of the loci tested. When the analysis results were inspected, only for 2 of these had a failed/discordant result, while for the remaining 5 the homozygous typing result had passed the QC tests. The investigation focused on the amplification step: the thermocyclers were checked, the reagents had already been in use with no issues, and only the polypropylene 96-well plates used were different, as we had just switched to plates from another manufacturer. The plates previously used were re-ordered and the discordant pairs were repeated using them. The amplification step was successful and the typing results confirmed that in the 5 QC-passed discordant pairs, there was allelic loss. These results suggest that in that run, the conditions of the reactions were suboptimal and resulted in the preferential amplification of one allele. The importance of plasticware in the performance of advanced methods comprising a big number of steps, can be overlooked in the biggest picture. But the effect can be important and sometimes not immediately recognizable, as was the case here where in the initial reactions the affected samples had a seemingly normal amplification and passed all the quality metrics.