RESUMEN
Genomic sequencing of HLA-A*30:02:01:03, an intronic variant, and HLA-C*08:113, an exonic variant.
Asunto(s)
Alelos , Antígenos HLA-A/genética , Antígenos HLA-C/genética , Mutación , Sistema de Registros , Sustitución de Aminoácidos , Secuencia de Bases , Trasplante de Médula Ósea , Exones , Genotipo , Antígenos HLA-A/inmunología , Antígenos HLA-C/inmunología , Prueba de Histocompatibilidad , Humanos , Intrones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Alineación de Secuencia , Análisis de Secuencia de ADN , Sudáfrica , Donantes de TejidosRESUMEN
The hyperpolymorphic HLA genes play important roles in disease and transplantation and act as genetic markers of migration and evolution. A panel of 107 B-lymphoblastoid cell lines (B-LCLs) was established in 1987 at the 10th International Histocompatibility Workshop as a resource for the immunogenetics community. These B-LCLs are well characterised and represent diverse ethnicities and HLA haplotypes. Here we have applied Pacific Biosciences' Single Molecule Real-Time (SMRT) DNA sequencing to HLA type 126 B-LCL, including the 107 International HLA and Immunogenetics Workshop (IHIW) cells, to ultra-high resolution. Amplicon sequencing of full-length HLA class I genes (HLA-A, -B and -C) and partial length HLA class II genes (HLA-DRB1, -DQB1 and -DPB1) was performed. We typed a total of 931 HLA alleles, 895 (96%) of which were consistent with the typing in the IPD-IMGT/HLA Database (Release 3.27.0, January 20, 2017), with 595 (64%) typed at a higher resolution. Discrepant types, including novel alleles (n = 10) and changes in zygosity (n = 13), as well as previously unreported types (n = 34) were observed. In addition, patterns of linkage disequilibrium were distinguished by four-field resolution typing of HLA-B and HLA-C. By improving and standardising the HLA typing of these B-LCLs, we have ensured their continued usefulness as a resource for the immunogenetics community in the age of next generation DNA sequencing.
Asunto(s)
Sistemas de Computación , Antígenos HLA/genética , Inmunogenética , Internacionalidad , Análisis de Secuencia de ADN , Imagen Individual de Molécula , Alelos , Línea Celular , Humanos , Desequilibrio de Ligamiento/genéticaRESUMEN
Recent years have seen a rapid increase in the discovery of novel allelic variants of the human leukocyte antigen (HLA) genes. Commonly, only the exons encoding the peptide binding domains of novel HLA alleles are submitted. As a result, the IPD-IMGT/HLA Database lacks sequence information outside those regions for the majority of known alleles. This has implications for the application of the new sequencing technologies, which deliver sequence data often covering the complete gene. As these technologies simplify the characterization of the complete gene regions, it is desirable for novel alleles to be submitted as full-length sequences to the database. However, the manual annotation of full-length alleles and the generation of specific formats required by the sequence repositories is prone to error and time consuming. We have developed TypeLoader to address both these facets. With only the full-length sequence as a starting point, Typeloader performs automatic sequence annotation and subsequently handles all steps involved in preparing the specific formats for submission with very little manual intervention. TypeLoader is routinely used at the DKMS Life Science Lab and has aided in the successful submission of more than 900 novel HLA alleles as full-length sequences to the European Nucleotide Archive repository and the IPD-IMGT/HLA Database with a 95% reduction in the time spent on annotation and submission when compared with handling these processes manually. TypeLoader is implemented as a web application and can be easily installed and used on a standalone Linux desktop system or within a Linux client/server architecture. TypeLoader is downloadable from http://www.github.com/DKMS-LSL/typeloader.