Abstract:Sequencing‐based HLA typing (SBT) is a PCR based high resolution HLA typing method in which polymorphic regions of the gene are sequenced and directly used for typing. Currently, for class II SBT, alleles are identified by comparison of the exon 2 sequence with their corresponding allele sequence library. Routine SBT requires reliable identification of heterozygosity, and automated assignment of the alleles. In sequencing strategies different enzymes can be used for primer extension. The most characteristic difference between sequences obtained by two protocols using Sequenase®, or Taq‐cycle sequencing, respectively, is a difference in incorporation of nucleotides in the primer extension leading to different sequence profiles. In Taq‐cycling sequencing variable nucleotide incorporation results in irregular, but reproducible peak patterns, whereas Sequenase® incorporates nucleotides in nearly equal amounts, resulting in more even peak patterns. In a previously published multi‐center study we evaluated HLA‐DPB1 SBT using Taq‐cycle sequencing, and showed that typing can reliably be performed, considering the specific sequence profiles. In this study the applicability of Sequenase® for HLA‐DPB1 SBT was tested. A panel of samples were typed by SBT at five test sites which participate in the Sequencing Based Typing component of the 12thInternational Histocompatibility Workshop. The panel represents the existing polymorphism at all known polymorphic positions of exon 2, both in homozygous and heterozygous combinations. The assignment of homozygosity and heterozygosity was validated by Multi‐Sequence Analysis, performing cluster analysis of chromatographic data of all sequences at each position. Sequence characteristics were examined and considered for appropriate assignment. Data reveals that Sequenase® sequencing can also reliably be used