Typing For HLA-DRB3,DRB4 And DRB5 From Next-generation Sequencing Data

Human leukocyte antigen(HLA)molecules play an essential role in human immune system.HLA typing is the major way to study HLA gene variations.HLA typing method can be applied in organ or stem cell transplantation,population genetics,and study of autoimmune diseases,infectious diseases,cancer and other diseases.Along with the decrease of cost and increase of accuracy of next-generation sequencing(NGS),HLA typing based on NGS has been the major trend.Lots of HLA typing methods have been developed based on NGS,but few include DRB3/4/5.Through combination of reads mapping,assembly,and copy number dertermination by sequencing depth,typing for DRB3/4/5 can reach higher accuracy.A new DRB3/4/5 typing method is developed in this paper.Simulated and real NGS data are used to verify the accuracy of this method.A population genetics study and a disease study are conducted to display the applications of this method.Detailed research of this paper is listed below:1.Development of a new DRB3/4/5 typing method based on NGS data.New method relies on database of types.It aligns reads to reference sequenc and assembly them to haplotypes.Haplotypes are filtered,scored and used to predict HLA types.Copy number of DRB3/4/5 is also determined.Simulated NGS data is used to evaluate the new method,and 100%accuracy is reached at 4-digit resolution with 30-fold coverage.Real NGS data of 188 samples is also used for evaluation,and accuracy of DRB3/4/5 is 96.56%,98.89%and 99.34%at 4-digit resolution with 40-fold coverage.2.New method is applied in a population genetics study.Whole genome sequencing(WGS)data of 150 samples from 5 different populations in 1000 genome project are analysed.Frequency of DRB4 is significantly different between african and other populations.Linkage disequilibrium between DRB1 and DRB3/4/5 is consistent with common knowledge about haplotype construction of DRB genes.3.New method is applied in a disease study.100 patients with PLA2R-related membranous nephropathy(MN),50 patients with PLA2R-unrelated MN and 100 healthy controls are included.All samples are sequenced by MHC capture array and NGS.DRB1*15:01 and DRB3*02:02 are found to be independent risk alleles of PLA2R-related MN.DRB3*02:02 is identified as independent risk allele of PLA2R-unrelated MN.