Application of high-resolution, massively parallel pyrosequencing for estimation of haplotypes and gene expression levels of swine leukocyte antigen (SLA) class I genes
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The swine is an important animal model for allo- and xeno-transplantation donor studies, which necessitates an extensive characterization of the expression and sequence variations within the highly polygenic and polymorphic swine leukocyte antigen (SLA) region. Massively parallel pyrosequencing is potentially an effective new 2ndGen method for simultaneous high-throughput genotyping and detection of SLA class I gene expression levels. In this study, we compared the 2ndGen method using the Roche Genome Sequencer 454 FLX with the conventional method using sub-cloning and Sanger sequencing to genotype SLA class I genes in five pigs of the Clawn breed and four pigs of the Landrace breed. We obtained an average of 10.4 SLA class I sequences per pig by the 2ndGen method, consistent with the inheritance data, and an average of only 6.0 sequences by the conventional method. We also performed a correlation analysis between the sequence read numbers obtained by the 2ndGen method and the relative expression values obtained by quantitative real-time PCR analysis at the allele level. A significant correlation coefficient (r = 0.899, P < 0.01) was observed between the sequence read numbers and the relative quantitative values for the expressed classical SLA class I genes SLA-1, SLA-2, and SLA-3, suggesting that the sequence read numbers closely reflect the gene expression levels in white blood cells. Overall, five novel class I sequences, different haplotype-specific expression patterns and a splice variant for one of the SLA class I genes were identified by the 2ndGen method at greater efficiency and sensitivity than the conventional method.
KeywordsSwine MHC Genotyping Haplotype Real-time PCR Pyrosequencing
We thank the Japan Farm CLAWN Institute (Kagoshima, Japan) and the National Institute of Livestock and Grassland Sciences (Tsukuba, Japan) for providing the swine blood samples. We thank Masayuki Tanaka, Hideki Hayashi, and Hiroshi Kamiguchi in Education and Research Support Center in the Tokai University School of Medicine for technical assistance. The work was supported by Grant-in-Aid for Scientific Research on Innovative Areas (22133002) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), Grant-in-Aid for Scientific Research (B) (21300155) from Japan Society for the Promotion of Science (JSPS) and the Animal Genome Research Project of the Ministry of Agriculture, Forestry and Fisheries of Japan.
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