MULTI LOCUS SEQUENCING BASED TYPING: ITS POTENTIAL AS A NOVEL HLA SEQUENCING BASED TYPING TECHNIQUE.   David Sayer^1,2, Erik Rozemuller³, Joseph Meier¹, Campbell Witt¹, Marcel Tilanus³ and Frank Christiansen^1,2. ¹Department of Clinical Immunology, Royal Perth Hospital, Western Australia. ²Department of Pathology, University of Western Australia, ³Department of Pathology, University Hospital, Utrecht, The Netherlands.


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DNA Sequencing Based Typing (SBT) provides high resolution typing for HLA-A, -B and -C but is too cumbersome to be the single, routine method for HLA Class I typing in high throughput typing laboratories because each loci is amplified and sequenced independantly. Therefore, we have investigated the utility of a novel SBT procedure where the 3 genes are sequenced simultaneously. This procedure is called Multi Locus Sequencing Based Typing (MLSBT). As a preliminary evaluation of this technique we designed computer software programs to simulate MLSBT on a panel of 50 genotypes to determine the expected typing resolution. First, the software determines the multi locus sequence (MLS) for each of the genotypes and then identifies which other genotypes from a complete list of HLA alleles would result in the same MLS as the test. The resulting list then enables the degree of typing resolution expected for the test genotype to be determined. Two methods of calculating the MLS were compared. One was qualitative and describes only the nucleotides present at each site. The other method is quantitative and considers the relative amounts of each nucleotide at each site. . From the analysis of 50 genotypes, the typing resolution by qualitative analysis for each allele depends on the other alleles present but can provide high resolution typing for some loci in many instances and provide adequate resolution for many clinical requirements. Quantitative analysis provides high resolution typing to level expected if each locus was sequenced independantly, indicating that quantitative MLSBT has the potential to provide high resolution typing for HLA-A, -B and -C by sequencing a single template. (9807)