title: An integrated genotyping approach for HLA and other complex genetic systems.
abstract: Clinical immunogenetics laboratories performing routine sequencing of human leukocyte antigen (HLA) genes in support of hematopoietic cell transplantation are motivated to upgrade to next-generation sequencing (NGS) technology by its potential for cost savings as well as testing accuracy and flexibility. While NGS machines are available and simple to operate, there are few systems available that provide comprehensive sample preparation and data analysis workflows to complete the process. We report on the development and testing of the Integrated Genotyping System (IGS), which has been designed to specifically address the challenges associated with the adoption of NGS in clinical laboratories. To validate the system for a variety of sample DNA sources, we have tested 336 DNA specimens from whole blood, dried blood spots, buccal swabs, and lymphoblastoid cell lines. HLA class I and class II genotypes were derived from amplicon sequencing of HLA-A, -B, -C for exons 1-7 and HLA-DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4, -DRB5 for exons 1-4. Additionally, to demonstrate the extensibility of the IGS to other genetic loci, KIR haplotyping of 93 samples was carried out in parallel with HLA typing using a workflow based on the HLA system. These results are discussed with respect to their applications in the clinical setting and consequent potential for advancing precision medicine.
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States; Scisco Genetics Inc., Seattle, WA 98115, United States.
authors.forename: Wyatt C
authors.initials: WC
authors.lastname: Nelson
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States; Scisco Genetics Inc., Seattle, WA 98115, United States.
authors.forename: Chul-Woo
authors.initials: CW
authors.lastname: Pyo
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States; Scisco Genetics Inc., Seattle, WA 98115, United States.
authors.forename: David
authors.initials: D
authors.lastname: Vogan
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States; Scisco Genetics Inc., Seattle, WA 98115, United States.
authors.forename: Ruihan
authors.initials: R
authors.lastname: Wang
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States.
authors.forename: Yoon-Soo
authors.initials: YS
authors.lastname: Pyon
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States.
authors.forename: Carly
authors.initials: C
authors.lastname: Hennessey
authors.affiliation: Scisco Genetics Inc., Seattle, WA 98115, United States.
authors.forename: Anajane
authors.initials: A
authors.lastname: Smith
authors.affiliation: Scisco Genetics Inc., Seattle, WA 98115, United States.
authors.forename: Shalini
authors.initials: S
authors.lastname: Pereira
authors.affiliation: Scisco Genetics Inc., Seattle, WA 98115, United States; Scisco Genetics Inc., Shinga-cho, Kashihara, Nara 634-0006, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Ishitani
authors.affiliation: Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States; Scisco Genetics Inc., Seattle, WA 98115, United States; Scisco Genetics Inc., Shinga-cho, Kashihara, Nara 634-0006, Japan. Electronic address: geraghty@fhcrc.org.
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Geraghty
mesh_terms: D000483:Alleles; D019295:Computational Biology / Q000379:methods; D005091:Exons; D005838:Genotype; D060005:Genotyping Techniques; D006680:HLA Antigens / Q000235:genetics*; D006239:Haplotypes; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods* / Q000592:standards; D006650:Histocompatibility Testing; D006801:Humans; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D012984:Software; D057188:Workflow
chemical_list: D006680:HLA Antigens
keywords: Genotyping software; HLA genotyping; High resolution; KIR haplotyping; Multiplex PCR; Single molecule sequencing; System integration