Next-generation sequencing: the solution for high-resolution, unambiguous human leukocyte antigen typing.
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.
Lind
C
C
Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Ferriola
D
D
Mackiewicz
K
K
Heron
S
S
Rogers
M
M
Slavich
L
L
Walker
R
R
Hsiao
T
T
McLaughlin
L
L
D'Arcy
M
M
Gai
X
X
Goodridge
D
D
Sayer
D
D
Monos
D
D
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
D017931:DNA Primers