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Related Articles Rapid High-throughput HLA Typing by Massively-parallel Pyrosequencing for High-Resolution Allele Identification.
Hum Immunol. 2009 Aug 22;
Authors: Gabriel C, Danzer M, Hackl C, Kopal G, Hufnagl P, Hofer K, Polin H, Stabentheiner S, Pröll J
Transplantation and notably haematopoietic stem cell transplantation require high resolution HLA typing and are therefore, because of the heterozygous genomic DNA samples, dependent on clonal analytical methods. High-resolution HLA typing is a necessity for accomplishing the best-possible histocompatibility match between donor and recipient, since 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, human leukocyte antigen 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, USA). 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 adaptors were lengthened with donor-identifying barcode sequences to identify each of 8 Caucasian reference donors within one single multiplex sequencing run. Results, compared to normal SBT HLA typing show that with an average of 1000 reads per amplicon every patient was identified correctly. 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.
PMID: 19706315 [PubMed - as supplied by publisher]
More...
Related Articles Rapid High-throughput HLA Typing by Massively-parallel Pyrosequencing for High-Resolution Allele Identification.
Hum Immunol. 2009 Aug 22;
Authors: Gabriel C, Danzer M, Hackl C, Kopal G, Hufnagl P, Hofer K, Polin H, Stabentheiner S, Pröll J
Transplantation and notably haematopoietic stem cell transplantation require high resolution HLA typing and are therefore, because of the heterozygous genomic DNA samples, dependent on clonal analytical methods. High-resolution HLA typing is a necessity for accomplishing the best-possible histocompatibility match between donor and recipient, since 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, human leukocyte antigen 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, USA). 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 adaptors were lengthened with donor-identifying barcode sequences to identify each of 8 Caucasian reference donors within one single multiplex sequencing run. Results, compared to normal SBT HLA typing show that with an average of 1000 reads per amplicon every patient was identified correctly. 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.
PMID: 19706315 [PubMed - as supplied by publisher]
More...