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

被引:109
作者
Lind, C. [1 ]
Ferriola, D. [1 ]
Mackiewicz, K. [1 ]
Heron, S. [1 ]
Rogers, M. [1 ]
Slavich, L. [1 ]
Walker, R. [1 ]
Hsiao, T. [1 ]
McLaughlin, L. [1 ]
D'Arcy, M. [2 ]
Gai, X. [2 ]
Goodridge, D. [3 ]
Sayer, D. [3 ]
Monos, D. [1 ,4 ]
机构
[1] Childrens Hosp Philadelphia, Dept Pathol & Lab Med, Philadelphia, PA 19104 USA
[2] Childrens Hosp Philadelphia, Bioinformat Core Facil, Philadelphia, PA 19104 USA
[3] Conexio Genom, Fremantle, WA, Australia
[4] Univ Penn, Dept Pathol & Lab Med, Philadelphia, PA 19104 USA
关键词
HLA polymorphism; Ambiguities; DNA sequencing; High-resolution HLA typing; MAJOR HISTOCOMPATIBILITY COMPLEX; BONE-MARROW-TRANSPLANTATION; HLA-IDENTICAL SIBLINGS; MISMATCHES; LEUKEMIA; IMPACT; SITE; OUTCOMES; DONORS; ALLELE;
D O I
10.1016/j.humimm.2010.06.016
中图分类号
R392 [医学免疫学]; Q939.91 [免疫学];
学科分类号
100102 ;
摘要
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. (C) 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
引用
收藏
页码:1033 / 1042
页数:10
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