NOVEL AMBIGUITY RESOLUTION FOR HLA SBT METHODS.

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NOVEL AMBIGUITY RESOLUTION FOR HLA SBT METHODS.
Steven Cate A.S. , Runying Tian M.S. , MaKenzie Roberts B.S. and William Hildebrand Ph.D. . ¹ Microbiology and Immunology, University of Oklahoma Health Science Center, Oklahoma City, OK, USA .

The Major Histocompatibility Complex (MHC) is recognized as the most polymorphic region of the human genome. Our Clinical HLA Typing Lab at the University of Oklahoma Health Sciences Center has performed over 17,000 Class I typings and 11,000 Class II typings in the past 8 years. In 2004, we procured an ABI 3700 that we have successfully sustained with a daily work load of 768 sequencing reactions for time constrained contracts. We have one by one identified and overcome various capacity constraints at points in the HLA Typing production process. The final capacity constraints are the numerous and diverse ambiguities that require resolution. Group Specific Sequencing (GSS) can be used to resolve ambiguous combinations resulting from certain allelic combinations of heterozygous sequence which arise in an average of 21% of Class I (n=1500) and 15% of DRB1 samples (n=500). GSS compared to the commonly used Group Specific Amplification (GSA) is an incredibly simple and reliable method with considerable cost savings. GSS will resolve a majority of ambiguities that arise from the combination of the 400 DRB1 different alleles due to a problematic ambiguity near the end of Exon 2. We have also used this technique to eliminate Group Specific Amplification steps in our DQA1 typing strategy. The final application is quick confirmation or elimination of Potentially New Alleles by analysis of homozygous sequence produced by GSS. The entire primary typing and secondary ambiguity resolution is accomplished with a single DNA sequencing platform and no additional PCR steps.