SEQUENCE-BASED TYPING OF HLA CLASS II DRB1, B3, B4 and B5.
D Nuesca, P Chretien, D Eckels*, M Hessner*, B Luhm*, CE Garey. Visible Genetics, Inc., Toronto, ON, Canada; The Blood Center of Southeastern Wisconsin, Inc., Milwaukee, WI, USA. 

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As sequence-based-typing (SBT) has the ability to detect existing as well as new HLA polymorphisms, it is recognized as the current gold standard for high resolution HLA analysis. We have therefore developed a SBT-based methodology to provide for the rapid and precise typing of HLA Class II DRB1, B3, B4 and B5 loci using the Visible Genetics, Inc. OpenGene system. For SBT, all samples were first PCR-amplified with group-specific DRB1, B3, B4 and B5 primers. The amplification products were then sequenced simultaneously in both directions with dye-labelled forward and reverse primers by CLIP sequencing. The sequence data was generated by a 30 minute run on the MicroGene Clipper Sequencer. Data was analysed and typed with the GeneLibrarian software for HLA typing. This SBT method was subjected to testing by external DNA typing laboratories as wells as internally at Visible Genetics, Inc. The testing used samples obtained from routine batches as well as samples prepared from cell lines. These results were then compared to results obtained with the same samples using other typing methods. The samples used in the testing program have been found to be representative of the most frequently observed antigens and indicate excellent concordance with other typing methods. This robust HLA Class II SBT methodology can be routinely utilised for matching donor-recipient pairs prior to transplantation and in disease association or immune response studies where precise knowledge of HLA Class II sequence variation is demanded.