Rapid high-throughput human leukocyte antigen typing by massively parallel pyrosequencing for high-resolution allele identification.

Transplantation and, notably, hematopoietic stem cell transplantation require high-resolution human leukocyte antigen (HLA) typing and, because of the heterozygous genomic DNA samples, are dependent on clonal analytical methods. High-resolution HLA typing is a necessity for accomplishing the best possible histocompatibility match between donor and recipient, because mismatches strongly increase the risk of severe acute graft-versus-host disease. We describe the development and first application in a clinical setting of a novel, HLA sequence-based typing method by exploring the next-generation sequencing technology as provided by the Genome Sequencer FLX system (Roche/454 Life Sciences, Branford, CT). The developed system allows for ambiguity-free, high-throughput, high-resolution HLA-A and -B typing with the potential for automation. Primers and Genome Sequencer FLX specific adapters were lengthened with donor-identifying barcode sequences to identify each of eight Caucasian reference donors within one single multiplex sequencing run. Compared with normal SBT HLA typing, results indicate that every patient was identified correctly with an average of 1000 reads per amplicon. Furthermore, current investments for increased read lengths and fully automated molecular diagnostic software tools, using original GS-FLX data file formats, will enhance this novel HLA typing strategy in the near future.