title: A multi-site study using high-resolution HLA genotyping by next generation sequencing.
abstract: The high degree of polymorphism at human leukocyte antigen (HLA) class I and class II loci makes high-resolution HLA typing challenging. Current typing methods, including Sanger sequencing, yield ambiguous typing results because of incomplete genomic coverage and inability to set phase for HLA allele determination. The 454 Life Sciences Genome Sequencer (GS FLX) next generation sequencing system coupled with conexio atf software can provide very high-resolution HLA genotyping. High-throughput genotyping can be achieved by use of primers with multiplex identifier (MID) tags to allow pooling of the amplicons generated from different individuals prior to sequencing. We have conducted a double-blind study in which eight laboratory sites performed amplicon sequencing using GS FLX standard chemistry and genotyped the same 20 samples for HLA-A, -B, -C, DPB1, DQA1, DQB1, DRB1, DRB3, DRB4, and DRB5 (DRB3/4/5) in a single sequencing run. The average sequence read length was 250 base pairs and the average number of sequence reads per amplicon was 672, providing confidence in the allele assignments. Of the 1280 genotypes considered, assignment was possible in 95% of the cases. Failure to assign genotypes was the result of researcher procedural error or the presence of a novel allele rather than a failure of sequencing technology. Concordance with known genotypes, in cases where assignment was possible, ranged from 95.3% to 99.4% for the eight sites, with overall concordance of 97.2%. We conclude that clonal pyrosequencing using the GS FLX platform and CONEXIO ATF software allows reliable identification of HLA genotypes at high resolution.
authors.affiliation: Roche Molecular Systems Inc. (RMS), Pleasanton, CA 94588, USA.
authors.forename: C L
authors.initials: CL
authors.lastname: Holcomb
authors.forename: B
authors.initials: B
authors.lastname: Höglund
authors.forename: M W
authors.initials: MW
authors.lastname: Anderson
authors.forename: L A
authors.initials: LA
authors.lastname: Blake
authors.forename: I
authors.initials: I
authors.lastname: Böhme
authors.forename: M
authors.initials: M
authors.lastname: Egholm
authors.forename: D
authors.initials: D
authors.lastname: Ferriola
authors.forename: C
authors.initials: C
authors.lastname: Gabriel
authors.forename: S E
authors.initials: SE
authors.lastname: Gelber
authors.forename: D
authors.initials: D
authors.lastname: Goodridge
authors.forename: S
authors.initials: S
authors.lastname: Hawbecker
authors.forename: R
authors.initials: R
authors.lastname: Klein
authors.forename: M
authors.initials: M
authors.lastname: Ladner
authors.forename: C
authors.initials: C
authors.lastname: Lind
authors.forename: D
authors.initials: D
authors.lastname: Monos
authors.forename: M J
authors.initials: MJ
authors.lastname: Pando
authors.forename: J
authors.initials: J
authors.lastname: Pröll
authors.forename: D C
authors.initials: DC
authors.lastname: Sayer
authors.forename: G
authors.initials: G
authors.lastname: Schmitz-Agheguian
authors.forename: B B
authors.initials: BB
authors.lastname: Simen
authors.forename: B
authors.initials: B
authors.lastname: Thiele
authors.forename: E A
authors.initials: EA
authors.lastname: Trachtenberg
authors.forename: D B
authors.initials: DB
authors.lastname: Tyan
authors.forename: R
authors.initials: R
authors.lastname: Wassmuth
authors.forename: S
authors.initials: S
authors.lastname: White
authors.forename: H A
authors.initials: HA
authors.lastname: Erlich
mesh_terms: D000483:Alleles; D001483:Base Sequence; D004311:Double-Blind Method; D005191:Family Characteristics; D005838:Genotype; D006680:HLA Antigens / Q000032:analysis / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D015337:Multicenter Studies as Topic; D017422:Sequence Analysis, DNA / Q000379:methods / Q000639:trends*; D012984:Software
chemical_list: D006680:HLA Antigens