Journal Article

High-throughput, high-fidelity HLA genotyping with deep sequencing

Davis, Mark M, Wilhelmy, Julie, Wang, Chunlin, Fernandez-Viña, Marcelo A, Davis, Ronald W, Krishnakumar, Sujatha, Su, Laura F, Stepanyan, Lilit, Mindrinos, Michael, Babrzadeh, Farbod, Levinson, Douglas

2012

Overview

Human leukocyte antigen (HLA) genes are the most polymorphic in the human genome. They play a pivotal role in the immune response and have been implicated in numerous human pathologies, especially autoimmunity and infectious diseases. Despite their importance, however, they are rarely characterized comprehensively because of the prohibitive cost of standard technologies and the technical challenges of accurately discriminating between these highly related genes and their many allelles. Here we demonstrate a high-resolution, and cost-effective methodology to type HLA genes by sequencing, which combines the advantage of long-range amplification, the power of high-throughput sequencing platforms, and a unique genotyping algorithm. We calibrated our method for HLA-A, -B, -C, and -DRB1 genes with both reference cell lines and clinical samples and identified several previously undescribed alleles with mismatches, insertions, and deletions. We have further demonstrated the utility of this method in a clinical setting by typing five clinical samples in an Illumina MiSeq instrument with a 5-d turnaround. Overall, this technology has the capacity to deliver low-cost, high-throughput, and accurate HLA typing by multiplexing thousands of samples in a single sequencing run, which will enable comprehensive disease-association studies with large cohorts. Furthermore, this approach can also be extended to include other polymorphic genes.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: Algorithms / Alleles / antigens / autoimmunity / Base Sequence / Biological Sciences / Cell Line / DNA Primers / DNA Primers - genetics / Exons / Genes / genetics / Genomes / Genomics / Genotype / Genotype & phenotype / Genotypes / genotyping / Genotyping Techniques / Genotyping Techniques - methods / hematopoietic stem cell transplantation / high-throughput nucleotide sequencing / High-Throughput Nucleotide Sequencing - methods / Histocompatibility testing / HLA A antigens / HLA Antigens / HLA Antigens - genetics / HLA B antigens / HLA-A Antigens - genetics / HLA-B Antigens - genetics / HLA-C Antigens / HLA-C Antigens - genetics / HLA-DRB1 Chains / HLA-DRB1 Chains - genetics / Humans / Immune response / Immunity (Disease) / Infectious diseases / methods / Molecular Sequence Data / Polymerase Chain Reaction / Polymerase Chain Reaction - methods / Polymorphism / Polymorphism, Genetic / Reproducibility of Results / Sequence Homology, Nucleic Acid / sequence-based typing / Sequencing