AN EVALUATION OF A MULTICENTER STUDY ON HLA-DPB1 TYPING USING SOLID-PHASE TAQ-CYCLE SEQUENCING CHEMISTRY

In HLA Class II genes, polymorphism is mainly located in the second ex on, Most DNA based typing methods are confined to the identification o f specific sequence motifs in the second exon. In contrast, Sequencing Based Typing (SBT) elucidates the entire exon 2 sequence for typing. Comparison of the obtained exon 2 sequence with an allele sequence lib rary results in allele assignment. We tested the applicability of SET using a protocol for amplification followed by solid phase Tag-cycle s equencing for HLA-DPB 1 typing. A panel of 32 samples were typed by SB T at live test sites which are participating in the Sequencing Based T yping component of the 12th International Histocompatibility Workshop. The panel represents the existing polymorphism at all known polymorph ic positions of exon 2, both in homozygous and heterozygous combinatio ns. In this multicenter study we focused on the reliability of analyzi ng heterozygous sequences for HLA typing. A multi-sequence analysis ap proach, Polall, was developed to evaluate sequences obtained. The assi gnment of homozygosity and heterozygosity was validated by cluster ana lysis of chromatographic data of all sequences. Sequence characteristi cs were examined and considered for appropriate assignment. Difference s in sequence characteristics that occurred between the test sites are considered in detail. The evaluation of data of 5 test sites reveals that Tag-cycle sequencing can reliably be performed for HLA-DPB1 SBT.