High-sensitivity HLA Typing By Saturated Tiling Capture Sequencing(STC-Seq)

Background:The human leukocyte antigen(HLA)genes with a high degree of polymorphism is the most complex system in human gene at present.Its location occupies only 0.13%of the human genome,but it is associated with more than 100 diseases,mainly in regulating immune response,immune dysfunction,disease susceptibility,cancer,and drug reactions.The genotyping of HLA genes can promote understanding of the causality of the disease and promote the development of transplantation medicine.High-resolution HLA typing results are important for the treatment of autoimmune diseases and infectious diseases.In the field of transplantation medicine,the high-resolution typing result of HLA gene is also extremely important.If the HLA matching between donor and recipient is not compatible,it will cause immune rejection after organ or bone marrow transplantation.The establishment of a large-scale HLA high-resolution typing database is also necessary for the field of transplantation medicine and precision medicine.Although many HLA typing approaches have been developed,the complexity,low-efficiency and high-cost of current HLA-typing assays limit their application in population-based high-throughput HLA typing for donors.The sequence length of the third-generation sequencing technology using single molecule Real-Time(SMRT)sequencing method can up to 20 kb,which enable to achieve super-high-resolution result of HLA typing.While PCR-based methods have potential risks such as chimerism or drop-out when obtaining libraries for third generation sequencing,the acquisition of high-quality libraries is critical to achieving super-high-resolution result of HLA typing.Results:Based on the above reasons,we present a cost-efficient Saturated Tiling Capture Sequencing(STC-Seq)to achieve HLA typing.The method captures DNA fragments of 14 HLA genes at one time,including HLA-A,HLA-B,HLA-C,HLA-E and HLA-G from the HLA class ?(HLA-?)and HLA-DPA1,HLA-DPB1,HLA-DQA1,HLA-DQB1,HLA-DRA,HLA-DRB1,HLA-DRB3,HLA-DRB4 and HLA-DRB5 from HLA class ?(HLA-?).We have also developed a corresponding HLA typing algorithm.By testing on five genes(HLA-A,HLA-B,HLA-C,HLA-DRB1 and HLA-DQB1)from 31 standard samples from the China Marrow Donor Program(CMDP)and 351 datasets,STC-Seq showed results that were 98%consistent with the known two sets of digitals(fieldl and field2)genotypes.Additionally,STC can capture genomic DNA fragments longer than 3 kb from HLA loci,making the library compatible with the third-generation sequencing.Conclusion:In summary,STC-Seq is a highly accurate and cost-effi cient method for HLA typing which can be used to facilitate the establishment of population-based HLA databases for the precision and transpl antation medicine.