Next-generation sequencing: the solution for high-resolution, unambiguous human leukocyte antigen typing.
Hum Immunol
; 71(10): 1033-42, 2010 Oct.
Article
en En
| MEDLINE
| ID: mdl-20603174
ABSTRACT
Human leukocyte antigen (HLA) typing has been a challenge for more than 50 years. Current methods (Sanger sequencing, sequence-specific primers [SSP], sequence-specific oligonucleotide probes [SSOP]) continue to generate ambiguities that are time-consuming and expensive to resolve. However, next-generation sequencing (NGS) overcomes ambiguity through the combination of clonal amplification, which provides on-phase sequence and a high level of parallelism, whereby millions of sequencing reads are produced enabling an expansion of the HLA regions sequenced. We explored HLA typing using NGS through a three-step process. First, HLA-A, -B, -C, -DRB1, and -DQB1 were amplified with long-range PCR. Subsequently, amplicons were sequenced using the 454 GS-FLX platform. Finally, sequencing data were analyzed with Assign-NG software. In a single experiment, four individual samples and two mixtures were sequenced producing >75 Mb of sequence from >300,000 individual sequence reads (average length, 244 b). The reads were aligned and covered 100% of the regions amplified. Allele assignment was 100% concordant with the known HLA alleles of our samples. Our results suggest this method can be a useful tool for complete genomic characterization of new HLA alleles and for completion of sequence for existing, partially sequenced alleles. NGS can provide complete, unambiguous, high-resolution HLA typing; however, further evaluation is needed to explore the feasibility of its routine use.
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Prueba de Histocompatibilidad
/
Análisis de Secuencia de ADN
Tipo de estudio:
Clinical_trials
/
Diagnostic_studies
Límite:
Humans
Idioma:
En
Revista:
Hum Immunol
Año:
2010
Tipo del documento:
Article
País de afiliación:
Estados Unidos