title: Next-generation sequencing: the solution for high-resolution, unambiguous human leukocyte antigen typing.
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.
authors.affiliation: Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
authors.forename: C
authors.initials: C
authors.lastname: Lind
authors.forename: D
authors.initials: D
authors.lastname: Ferriola
authors.forename: K
authors.initials: K
authors.lastname: Mackiewicz
authors.forename: S
authors.initials: S
authors.lastname: Heron
authors.forename: M
authors.initials: M
authors.lastname: Rogers
authors.forename: L
authors.initials: L
authors.lastname: Slavich
authors.forename: R
authors.initials: R
authors.lastname: Walker
authors.forename: T
authors.initials: T
authors.lastname: Hsiao
authors.forename: L
authors.initials: L
authors.lastname: McLaughlin
authors.forename: M
authors.initials: M
authors.lastname: D'Arcy
authors.forename: X
authors.initials: X
authors.lastname: Gai
authors.forename: D
authors.initials: D
authors.lastname: Goodridge
authors.forename: D
authors.initials: D
authors.lastname: Sayer
authors.forename: D
authors.initials: D
authors.lastname: Monos
mesh_terms: D017931:DNA Primers; D003951:Diagnostic Errors / Q000517:prevention & control; D005240:Feasibility Studies; D006650:Histocompatibility Testing / Q000379:methods / Q000639:trends*; D006801:Humans; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D017931:DNA Primers