title: Very high resolution single pass HLA genotyping using amplicon sequencing on the 454 next generation DNA sequencers: Comparison with Sanger sequencing.
abstract: Compared to Sanger sequencing, next-generation sequencing offers advantages for high resolution HLA genotyping including increased throughput, lower cost, and reduced genotype ambiguity. Here we describe an enhancement of the Roche 454 GS GType HLA genotyping assay to provide very high resolution (VHR) typing, by the addition of 8 primer pairs to the original 14, to genotype 11 HLA loci. These additional amplicons help resolve common and well-documented alleles and exclude commonly found null alleles in genotype ambiguity strings. Simplification of workflow to reduce the initial preparation effort using early pooling of amplicons or the Fluidigm Access Array™ is also described. Performance of the VHR assay was evaluated on 28 well characterized cell lines using Conexio Assign MPS software which uses genomic, rather than cDNA, reference sequence. Concordance was 98.4%; 1.6% had no genotype assignment. Of concordant calls, 53% were unambiguous. To further assess the assay, 59 clinical samples were genotyped and results compared to unambiguous allele assignments obtained by prior sequence-based typing supplemented with SSO and/or SSP. Concordance was 98.7% with 58.2% as unambiguous calls; 1.3% could not be assigned. Our results show that the amplicon-based VHR assay is robust and can replace current Sanger methodology. Together with software enhancements, it has the potential to provide even higher resolution HLA typing.
authors.affiliation: Stanford University, Palo Alto, CA, USA. Electronic address: fionay@stanford.edu.
authors.forename: F
authors.initials: F
authors.lastname: Yamamoto
authors.affiliation: Roche Molecular Systems (RMS), Pleasanton, CA, USA. Electronic address: bryan.hoglund@roche.com.
authors.forename: B
authors.initials: B
authors.lastname: Höglund
authors.affiliation: Stanford University, Palo Alto, CA, USA. Electronic address: marcelof@stanford.edu.
authors.forename: M
authors.initials: M
authors.lastname: Fernandez-Vina
authors.affiliation: Stanford University, Palo Alto, CA, USA. Electronic address: dtyan@stanford.edu.
authors.forename: D
authors.initials: D
authors.lastname: Tyan
authors.affiliation: Roche Molecular Systems (RMS), Pleasanton, CA, USA. Electronic address: melinda.rastrou@roche.com.
authors.forename: M
authors.initials: M
authors.lastname: Rastrou
authors.affiliation: Roche Molecular Systems (RMS), Pleasanton, CA, USA.
authors.forename: T
authors.initials: T
authors.lastname: Williams
authors.affiliation: Roche Molecular Systems (RMS), Pleasanton, CA, USA. Electronic address: priscilla.moonsamy@roche.com.
authors.forename: P
authors.initials: P
authors.lastname: Moonsamy
authors.affiliation: Conexio Genomics, Perth, Australia. Electronic address: damian@conexio-genomics.com.
authors.forename: D
authors.initials: D
authors.lastname: Goodridge
authors.affiliation: Stanford University, Palo Alto, CA, USA. Electronic address: matthew.anderson@BCW.edu.
authors.forename: M
authors.initials: M
authors.lastname: Anderson
authors.affiliation: Roche Molecular Systems (RMS), Pleasanton, CA, USA. Electronic address: herlich@chori.org.
authors.forename: H A
authors.initials: HA
authors.lastname: Erlich
authors.affiliation: Roche Molecular Systems (RMS), Pleasanton, CA, USA. Electronic address: cherie.holcomb@roche.com.
authors.forename: C L
authors.initials: CL
authors.lastname: Holcomb
mesh_terms: D000483:Alleles; D002460:Cell Line; D019295:Computational Biology / Q000379:methods; D006680:HLA Antigens / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods* / Q000592:standards; D006650:Histocompatibility Testing; D006801:Humans; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods / Q000592:standards; D012984:Software; D057188:Workflow
chemical_list: D006680:HLA Antigens
keywords: 454 GS FLX; DNA sequencing; HLA genotyping; NGS