143-P: ENHANCED HLA TYPING RESOLUTION WITH CELERA MOTIF-SPECIFIC SEQUENCING PRIMERS

Aim In a renewed effort to enhance the resolution power of Celera’s AlleleSEQR® HLA-SBT products, we initiated a project to design additional sequencing primers to resolve ambiguous HLA allele combinations. Methods Active market surveillance was conducted to identify ambiguities that could not be resolved with the currently available products. Reported allele combinations containing at least two common, well documented (CWD) alleles were analyzed using the HLA/IMGT database sequence alignment tool and searched for common cis/trans ambiguities across all loci. The results were applied to designing a set of new motif-specific ambiguity primers (MSPs), which were subsequently tested using the 3100, 3130, 3730 and 3500 platforms. Results The extended panel of MSPs was designed to utilize the smallest number of sequencing primers to resolve the maximum number of ambiguities across all class I loci by targeting common motifs rather than locus-specific sequences. Half of the reported class I ambiguities could be resolved with only 5 new motif-specific ambiguity primers functioning across multiple class I loci. Resolution for class I loci could be increased through an additional 9 primers targeting more locus-specific motifs. Four primers were designed for class II. We further tested the use of new MSPs with adjusted run conditions to allow sequencing though introns. Using this approach on a subset of reported allele combinations, together with novel designs to improve resolution in the proximity of primer annealing site, we were able to successfully resolve 90% of reported ambiguities. Conclusions We designed a new panel of motif-specific sequencing primers, which will increase the overall number of Celera HLA-SBT ambiguity resolving sequencing primers to a total of 53 for class I and class II HLA genes. This expanded panel of reagents is estimated to resolve the majority of allele combinations currently encountered in clinical labs. Kobrle: Celera: Employee; Stockholder. Bruce: Celera: Employee. Drews: Celera: Employee. Bost: Celera: Employee.