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dc.contributor.authorLank, Simon M
dc.contributor.authorGolbach, Brittney A
dc.contributor.authorCreager, Hannah M
dc.contributor.authorWiseman, Roger W
dc.contributor.authorKeskin, Derin Benerci
dc.contributor.authorReinherz, Ellis Leonard
dc.contributor.authorBrusic, Vladimir
dc.contributor.authorO’Connor, David H
dc.date.accessioned2013-04-26T17:25:06Z
dc.date.issued2012
dc.identifier.citationLank, Simon M, Brittney A Golbach, Hannah M Creager, Roger W Wiseman, Derin B Keskin, Ellis L Reinherz, Vladimir Brusic, and David H O’Connor. 2012. Ultra-high resolution hla genotyping and allele discovery by highly multiplexed cdna amplicon pyrosequencing. BMC Genomics 13: 378.en_US
dc.identifier.issn1471-2164en_US
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:10589781
dc.description.abstractBackground: High-resolution HLA genotyping is a critical diagnostic and research assay. Current methods rarely achieve unambiguous high-resolution typing without making population-specific frequency inferences due to a lack of locus coverage and difficulty in exon-phase matching. Achieving high-resolution typing is also becoming more challenging with traditional methods as the database of known HLA alleles increases. Results: We designed a cDNA amplicon-based pyrosequencing method to capture 94% of the HLA class I open-reading-frame with only two amplicons per sample, and an analogous method for class II HLA genes, with a primary focus on sequencing the DRB loci. We present a novel Galaxy server-based analysis workflow for determining genotype. During assay validation, we performed two GS Junior sequencing runs to determine the accuracy of the HLA class I amplicons and DRB amplicon at different levels of multiplexing. When 116 amplicons were multiplexed, we unambiguously resolved 99%of class I alleles to four- or six-digit resolution, as well as 100% unambiguous DRB calls. The second experiment, with 271 multiplexed amplicons, missed some alleles, but generated high-resolution, concordant typing for 93% of class I alleles, and 96% for DRB1 alleles. In a third, preliminary experiment we attempted to sequence novel amplicons for other class II loci with mixed success. Conclusions: The presented assay is higher-throughput and higher-resolution than existing HLA genotyping methods, and suitable for allele discovery or large cohort sampling. The validated class I and DRB primers successfully generated unambiguously high-resolution genotypes, while further work is needed to validate additional class II genotyping amplicons.en_US
dc.language.isoen_USen_US
dc.publisherBioMed Centralen_US
dc.relation.isversionofdoi:10.1186/1471-2164-13-378en_US
dc.relation.hasversionhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3575390/pdf/en_US
dash.licenseLAA
dc.subjectHLAen_US
dc.subjectGenotypingen_US
dc.subjectRoche/454en_US
dc.subjectPyrosequencingen_US
dc.subjectGalaxyen_US
dc.subjectTissue typingen_US
dc.subjectCellular immunityen_US
dc.subjectMultiplexingen_US
dc.titleUltra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencingen_US
dc.typeJournal Articleen_US
dc.description.versionVersion of Recorden_US
dc.relation.journalBMC Genomicsen_US
dash.depositing.authorReinherz, Ellis Leonard
dc.date.available2013-04-26T17:25:06Z
dc.identifier.doi10.1186/1471-2164-13-378*
dash.contributor.affiliatedBrusic, Vladimir
dash.contributor.affiliatedReinherz, Ellis
dash.contributor.affiliatedKeskin, Derin Benerci


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