Next‐generation sequencing of HLA: validation and identification of new polymorphisms in a Brazilian population

Abstract

BackgroundNext‐generation sequencing (NGS) is the most modern sequencing technique that has revolutionized HLA typing, providing high‐resolution results with low ambiguity rates. This study aimed to show the experiences and challenges of an HLA laboratory in the validation process of the NGS methodology for HLA typing and show the use of this method for the study of HLA genetic diversity.MethodsWe used 115 samples that comprised a comprehensive testing panel for validation of the NGS methodology using the AllType kit (One Lambda, Canoga Park, California) on the Ion Torrent S5 NGS platform. All quality metrics were analyzed. During validation, two new HLA sequences were identified and named by the HLA Nomenclature Committee.ResultsA total of 1380 alleles from the HLA‐A, ‐B, ‐C, ‐DRB1, ‐DQB1, and ‐DPB1 loci were examined by NGS. This validation panel provided a wide range of HLA sequence variations, including non‐CWD HLA alleles, new variants, and homozygous alleles. The concordance rate with Sanger sequencing‐based typing was 100.0% for HLA‐A, ‐B, ‐C, ‐DRB1, ‐DQB1, and 99.93% for HLA‐DPB1. The newly identified HLA alleles were HLA‐B*14:69N and HLA‐DQB1*02:145.ConclusionWe have successfully validated NGS HLA typing despite numerous challenges, contributing to the identification of novel alleles that impact on HLA matching and antibody evaluation in organ and tissue transplantation.

Identification of four novel HLA alleles: HLA‐B*27:265, ‐B*35:569, ‐DRB1*08:117 and ‐DPB1*1435:01

“…Informed consent was obtained from each donor. HLA typing was conducted using next‐generation sequencing (NGS) with the AllType™ kit (One Lambda, Inc., Canoga Park, California) on the Ion Torrent S5 platform (Thermo Fisher Scientific, Waltham, Massachusetts) according to the validated protocol 5 . Data was analyzed in Type Stream Visual software version 3.0.0 (One Lambda).…”

“…HLA typing was conducted using next-generation sequencing (NGS) with the AllType™ kit (One Lambda, Inc., Canoga Park, California) on the Ion Torrent S5 platform (Thermo Fisher Scientific, Waltham, Massachusetts) according to the validated protocol. 5 Data was analyzed in Type Stream Visual software version 3.0.0 (One Lambda). To confirm the new alleles, the Sequence-Based Typing (SBT) method was employed using the SeCore™HLA kit (One Lambda) and ABI 3730 platform (Applied Biosystems).…”

Identification of the novel HLA‐DQB1*04:85 allele by next‐generation sequencing

“…Next‐generation sequencing (NGS) methods are now increasingly employed for HLA genotyping in the Clinical HLA Laboratories, providing high‐resolution results with low ambiguity rates 1,2 . The number of HLA alleles is constantly increased with the application of molecular techniques.…”

The novel HLA‐DRB1*03:178, ‐DRB1*03:179, and ‐DRB1*11:276 alleles identified in a healthy Brazilian individuals

“…Approximately 8500 class II HLA alleles were officially recognized in the IPD‐IMGT/HLA Database (release 3.46, 2021‐10) 1 . The higher quality and reliability of HLA typing obtained by next‐generation sequencing of the complete HLA gene has contributed to improve the outcomes of hematopoietic stem cell transplantation (HSCT) 2,3 . In this report, we describe the full‐length sequence of three new HLA class II alleles in the healthy Brazilian individuals.…”