SEQUENASE SEQUENCE PROFILES USED FOR HLA-DPB1 SEQUENCING-BASED TYPING

Sequencing-based HLA typing (SET) is a PCR based high resolution HLA t yping method in which polymorphic regions of the gene are sequenced an d directly used for typing. Currently, for class II SET, alleles are i dentified by comparison of the exon 2 sequence with their correspondin g allele sequence library. Routine SET requires reliable identificatio n of heterozygosity, and automated assignment of the alleles. In seque ncing strategies different enzymes can be used for primer extension. T he most characteristic difference between sequences obtained by two pr otocols using Sequenase(R), or Tag-cycle sequencing? respectively, is a difference in incorporation of nucleotides in the primer extension l eading to different sequence profiles. In Tag-cycling sequencing varia ble nucleotide incorporation results in irregular, but reproducible pe ak patterns, whereas Sequenase(R) incorporates nucleotides in nearly e qual amounts, resulting in more even peak patterns. In a previously pu blished multi-center study we evaluated HLA-DPB1 SET using Tag-cycle s equencing, and showed that typing can reliably be performed, consideri ng the specific sequence profiles. In this study the applicability of Sequenase(R) for HLA-DPB1 SET was tested. A panel of samples were type d by SET at five test sites which participate in the Sequencing Based Typing component of the 12th International Histocompatibility Workshop . The panel represents the existing polymorphism at all known polymorp hic positions of exon 2, both in homozygous and heterozygous combinati ons. The assignment of homozygosity and heterozygosity was validated b y Multi-Sequence Analysis, performing cluster analysis of chromatograp hic data of all sequences at each position. Sequence characteristics w ere examined and considered for appropriate assignment. Data reveals t hat Sequenase(R) sequencing can also reliably be used for HLA-DPB1 typ ing.