A novel single cDNA amplicon pyrosequencing method for high-throughput, cost-effective sequence-based HLA class I genotyping.

Human Immunology
Simon M LankDavid H O'Connor

Abstract

Human leukocyte antigen (HLA) genotype influences the immune response to pathogens and transplanted tissues; accurate HLA genotyping is critical for clinical and research applications. Sequence-based HLA typing is limited by the cost of Sanger sequencing genomic DNA (gDNA) and resolving cis/trans ambiguities, hindering both studies correlating high-resolution genotype with clinical outcomes, and population-specific allele frequency surveys. We present an assay for sequence-based HLA genotyping by titanium read length clonal Roche/454 pyrosequencing of a single, universally diagnostic polymerase chain reaction (PCR) amplicon from HLA class I cDNA that captures most of exons 2, 3, and 4 used for traditional sequence-based typing. The amplicon is predicted to unambiguously resolve 85% of known alleles. A panel of 48 previously HLA-typed samples was assayed with this method, demonstrating 100% non-null allele typing concordance. We show that this technique can multiplex at least 768 patients per sequencing run with multiplex identifier sequence bar-coding. Unprecedented typing throughput results from a novel single cDNA-PCR amplicon strategy requiring only 1 PCR amplification per sample. This method dramatically reduces cost for ge...Continue Reading

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Citations

Nov 1, 2013·ILAR Journal·Roger W WisemanDavid H O'Connor
Mar 22, 2012·Nucleic Acids Research·Florent E AnglyGene W Tyson
Jan 20, 2011·BMC Genomics·Rachel L ErlichPaul I W de Bakker
May 30, 2013·BMC Genomics·Kazuyoshi HosomichiIturo Inoue
Jul 19, 2011·BMC Immunology·Justin M GreeneDavid H O'Connor
Dec 25, 2012·Genome Medicine·Sebastian BoegelUgur Sahin
Apr 5, 2013·PloS One·Steven RingquistMassimo Trucco
Jul 11, 2013·PloS One·Hyunsung John Kim, Nader Pourmand
Nov 14, 2013·PloS One·Endre MajorSzilveszter Juhos
Nov 16, 2013·Human Molecular Genetics·Ana Catarina AlvesMafalda Bourbon
Aug 22, 2014·Bioinformatics·András SzolekOliver Kohlbacher
Oct 2, 2015·Human Immunology·Jonathan C BaroneSusan Hsu
Jan 23, 2014·Tissue Antigens·C GabrielG F Fischer
Aug 28, 2015·Journal of Human Genetics·Kazuyoshi HosomichiIturo Inoue
Oct 5, 2014·Transfusion Medicine Reviews·Zhugong LiuRichard Davey
Jun 7, 2012·Transfusion Medicine and Hemotherapy : Offizielles Organ Der Deutschen Gesellschaft Fur̈ Transfusionsmedizin Und Immunham̈atologie·Christian GabrielJohannes Pröll
Aug 18, 2017·Journal of Extracellular Vesicles·Kenneth GouinAhmed Gamal-Eldin Ibrahim
Nov 2, 2019·Biology Direct·Chengsheng ZhuYana Bromberg

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