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Next-generation sequencing: the solution for high-resolution, unambiguous human leukocyte antigen typing

2010; Elsevier BV; Volume: 71; Issue: 10 Linguagem: Inglês

10.1016/j.humimm.2010.06.016

1879-1166

Curt Lind, Deborah Ferriola, Kate Mackiewicz, S. Heron, Marianne Rogers, Larissa Slavich, Russell Walker, Tom Hsiao, L. McLaughlin, Monica D’Arcy, Xiaowu Gai, Damian Goodridge, D. Sayer, Dimitri Monos,

Immunotherapy and Immune Responses

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.