Major histocompatibility complex genotyping with massively parallel pyrosequencing

Major histocompatibility complex (MHC) genetics dictate adaptive cellular immune responses, making robust MHC genotypin g methods essential for studies of infectious disease, vaccine development and transplantation. Nonhuman primates provide essential preclinic al models for these areas of biomedical research. Unfortunately, given the unparalleled complexity of macaque MHCs, existing methodologies ar e inadequate for MHC typing of these key model animals. Here we use pyrosequencing of complementary DNA–PCR amplicons as a general appro ach to determine comprehensive MHC class I genotypes in nonhuman primates. More than 500 unique MHC class I sequences were resolved by sequen ce-based typing of rhesus, cynomolgus and pig-tailed macaques, nearly half of which have not been reported previously. The remarkable sensiti vity of this approach in macaques demonstrates that pyrosequencing is viable for ultra–high-throughput MHC genotyping of primates, inclu ding humans.