title: HLA-genotyping of clinical specimens using Ion Torrent-based NGS.
abstract: We have evaluated and validated the NXType™ workflow (One Lambda, Inc.) and the accompanying TypeStream™ software on the Ion Torrent Next Generation Sequencing (NGS) platform using a comprehensive testing panel. The panel consisted of 285 genomic DNA (gDNA) samples derived from four major ethnic populations and contained 59 PT samples and 226 clinical specimens. The total number of alleles from the six loci interrogated by NGS was 3420. This validation panel provided a wide range of HLA sequence variations including many rare alleles, new variants and homozygous alleles. The NXType™ system (reagents and software) was able to correctly genotype the vast majority of these specimens. The concordance rate between SBT-derived genotypes and those generated by TypeStream™ auto-analysis ranged from 99.5% to 99.8% for the HLA-A, B, C, DRB1 and DQB1 loci, and was 98.9% for HLA-DPB1. A strategy for data review was developed that would allow correction of most of the few remaining typing errors. The entire NGS workflow from gDNA amplification to genotype assignment could be completed within 3 working days. Through this validation study, the limitations and shortcomings of the platform, specific assay system, and software algorithm were also revealed for further evaluation and improvement.
authors.affiliation: American Red Cross, 700 Spring Garden Street, Philadelphia, PA 19123, USA.
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Barone
authors.affiliation: One Lambda, Inc., 21001 Kittridge Street, Canoga Park, CA 91303, USA.
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Saito
authors.affiliation: One Lambda, Inc., 21001 Kittridge Street, Canoga Park, CA 91303, USA.
authors.forename: Karl
authors.initials: K
authors.lastname: Beutner
authors.affiliation: One Lambda, Inc., 21001 Kittridge Street, Canoga Park, CA 91303, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Campo
authors.affiliation: American Red Cross, 700 Spring Garden Street, Philadelphia, PA 19123, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Dong
authors.affiliation: Thomas Jefferson University Hospital, Molecular and Genomic Pathology Lab, 1025 Walnut Street, College Building, Suite 401, Philadelphia, PA 19107, USA.
authors.forename: Chirayu P
authors.initials: CP
authors.lastname: Goswami
authors.affiliation: Thomas Jefferson University Hospital, Molecular and Genomic Pathology Lab, 1025 Walnut Street, College Building, Suite 401, Philadelphia, PA 19107, USA.
authors.forename: Erica S
authors.initials: ES
authors.lastname: Johnson
authors.affiliation: Thomas Jefferson University Hospital, Molecular and Genomic Pathology Lab, 1025 Walnut Street, College Building, Suite 401, Philadelphia, PA 19107, USA.
authors.forename: Zi-Xuan
authors.initials: ZX
authors.lastname: Wang
authors.affiliation: American Red Cross, 700 Spring Garden Street, Philadelphia, PA 19123, USA. Electronic address: Susan.Hsu@redcross.org.
authors.forename: Susan
authors.initials: S
authors.lastname: Hsu
mesh_terms: D000483:Alleles; D019295:Computational Biology / Q000379:methods; D015723:Gene Library; D014644:Genetic Variation; D005838:Genotype; D006680:HLA Antigens / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods* / Q000592:standards; D006650:Histocompatibility Testing; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA
chemical_list: D006680:HLA Antigens
keywords: Genotyping; HLA class I, II; Isothermal amplification; Multiplex PCR; Next Generation Sequencing