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A novel single cDNA amplicon pyrosequencing method for high-throughput, cost-effective sequence-based HLA class-I genotyping.
Hum Immunol. 2010 Jul 19;
Authors: Lank SM, Wiseman RW, Dudley DM, O'Connor DH
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 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 PCR amplicon from HLA class-I cDNA that captures the majority 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 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 one PCR amplification per sample. This method dramatically reduces cost for genotyping of large cohorts.
PMID: 20650293 [PubMed - as supplied by publisher]
More...
A novel single cDNA amplicon pyrosequencing method for high-throughput, cost-effective sequence-based HLA class-I genotyping.
Hum Immunol. 2010 Jul 19;
Authors: Lank SM, Wiseman RW, Dudley DM, O'Connor DH
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 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 PCR amplicon from HLA class-I cDNA that captures the majority 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 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 one PCR amplification per sample. This method dramatically reduces cost for genotyping of large cohorts.
PMID: 20650293 [PubMed - as supplied by publisher]
More...