Tipu Hamid Nawaz
Department of Immunology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan.
J Coll Physicians Surg Pak. 2020 Nov;30(11):1138-1142. doi: 10.29271/jcpsp.2020.11.1138.
To compare two human leukocyte antigen (HLA) typing methods, namely sequence specific primers (SSP) and next generation sequencing (NGS) for alleles concordance Study Design: Descriptive study.
Immunology department, Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from December 2019 to May 2020.
A total of 48 subjects, including 30 males and 18 females, were typed by NGS at 7 loci, making a total of 672 types loci. SSP typing was done for 276 loci among these. Comparison was made at SSP level of low resolution. NGS typing was done with Illumina's MiSeq instrument using Omixon HLA holotype 7 loci kit and analysis done with HLA twin software. SSP typing was done with micro SSP kit from onelambda. Statistical analysis was done using statistical package for social sciences (SPSS) version 24.0.
Among the 672 NGS types loci and 276 SSP types loci, there were mismatches at one B locus and one C locus, whereby NGS computed HLA-B58:01 and HLA-C12:02 while SSP detected HLA-B57 and HLA-C05, respectively. At remaining 274 loci, HLA typing fully matched at low resolution, making concordance rate 99.3%. Commonest alleles detected by NGS were HLA-A02:01, B51:01, C07:02, DPB104:01, DQA101:03, DQB102:01 and DRB1*13:01.
Batch testing, high throughput, improved accuracy, more loci coverage, maximum gene coverage including all exons and introns and high-resolution typing confer significant advantages to next generation sequencing over old methods of HLA typing. This technique is suitable for high throughput laboratories. High running cost hampers its routine implementation in 3rd world countries. Key Words: Human leukocyte antigen, Next generation sequencing, High resolution.
比较两种人类白细胞抗原（HLA）分型方法，即序列特异性引物（SSP）和新一代测序（NGS），以研究等位基因一致性。研究设计：描述性研究。
2019年12月至2020年5月，位于巴基斯坦拉瓦尔品第的武装部队病理研究所免疫科。
共有48名受试者，包括30名男性和18名女性，通过NGS对7个位点进行分型，共获得672个位点分型。其中对276个位点进行了SSP分型。在低分辨率的SSP水平上进行比较。使用Illumina的MiSeq仪器，采用Omixon HLA全型7位点试剂盒进行NGS分型，并使用HLA twin软件进行分析。使用onelambda的微SSP试剂盒进行SSP分型。使用社会科学统计软件包（SPSS）24.0版进行统计分析。
在672个NGS分型位点和276个SSP分型位点中，在一个B位点和一个C位点出现不匹配，其中NGS计算出HLA-B58:01和HLA-C12:02，而SSP分别检测到HLA-B57和HLA-C05。在其余274个位点，低分辨率下HLA分型完全匹配，一致性率为99.3%。NGS检测到的最常见等位基因是HLA-A02:01、B51:01、C07:02、DPB104:01、DQA101:03、DQB102:01和DRB1*13:01。
批量检测、高通量、更高的准确性、更多的位点覆盖、包括所有外显子和内含子的最大基因覆盖以及高分辨率分型，使得新一代测序相对于旧的HLA分型方法具有显著优势。该技术适用于高通量实验室。高昂的运行成本阻碍了其在第三世界国家的常规应用。关键词：人类白细胞抗原；新一代测序；高分辨率
