A novel single cDNA amplicon pyrosequencing method for high-throughput, cost-effective sequence-based HLA class I genotyping.
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 genotyping of large cohorts.
Lank
Simon M
SM
Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Wiseman
Roger W
RW
Dudley
Dawn M
DM
O'Connor
David H
DH
D000483:Alleles; D003362:Cost-Benefit Analysis; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis*; D005805:Genes, MHC Class I / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000639:trends; D006650:Histocompatibility Testing / Q000191:economics; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000191:economics; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D018076:DNA, Complementary