부산대학교 도서관

Aim Recent advances in Next Generation sequencing technologies have enabled us to provide higher resolution typing for HLA in Antigen Recognition Sites (ARS) faster and more cost effectively compared to Sanger Sequencing. Using these technologies, we can also sequence whole gene and long range HLA Class I and II alleles that were amplified in one long amplicon (>3 kb). PacBio RSII platform’s Single molecule, real-time (SMRT®) sequencing, generating average long reads of 10–15 kb, allows us to obtain in-phase long sequence reads. We have introduced and applied the PacBio RSII platform in our lab for NMDP recruitment samples using whole gene (Class I) and long range (Class II) amplicons as sequencing templates. We present our findings of extensive variation beyond ARS especially in introns of Class I genes. Methods We performed whole gene Class I amplification on the genomic DNA samples using locus-specific primers at 5 ′ and 3 ′ UTRs. Whole gene products were sequenced on the PacBio RSII platform. In parallel, we performed Class I and Class II exon-based amplicon sequencing on the gDNA samples using the Illumina MiSeq platform. We analyzed the data based on 3.20.0 allele DB using our in house program. Results A total of 891 out of 3589 samples showed novel variations with 359 unique patterns. Most new variations were observed in introns of HLA-B, in particular intron 2 and 6 being the most active. Exon 6 showed the most novel variation among the exons having HLA-A the most. HLA-C did not have any new variation in exon 6 in this group. Only 4 novel alleles were observed in the ARS region, one in HLA-B and three in HLA-C. Most alleles that were named based on the ARS sequences had no or incomplete published sequences for the other regions of the gene at the completion of this study. Conclusion The whole gene sequencing of class I alleles is essential in determining the structural integrity and expressivity of the genes that could not be determined solely based on ARS. Intronic variation may be involved in modulation of the expression. Variation in intra cytoplasmic domain coding exons (Exons 6, 7, 8) may play a role in modulating signal transduction in Class I genes. Establishing a database with complete gene sequences will help further studies in clarifying the biological significance of those variations. S. Yang: Stock Shareholder; Company/Organization; PacBio . [ABSTRACT FROM AUTHOR]