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Ultra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing

Background: 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-reso... Full description

Journal Title: Lank 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.
Main Author: Lank, Simon M
Other Authors: Golbach, Brittney A , Creager, Hannah M , Wiseman, Roger W , Keskin, Derin Benerci , Reinherz, Ellis Leonard , Brusic, Vladimir , O’connor, David H
Format: Electronic Article Electronic Article
Language: English
Subjects:
Hla
ID: ISSN: 1471-2164 ; DOI: 10.1186/1471-2164-13-378
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recordid: dash1/10589781
title: Ultra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing
format: Article
creator:
  • Lank, Simon M
  • Golbach, Brittney A
  • Creager, Hannah M
  • Wiseman, Roger W
  • Keskin, Derin Benerci
  • Reinherz, Ellis Leonard
  • Brusic, Vladimir
  • O’connor, David H
subjects:
  • Hla
  • Genotyping
  • Roche/454
  • Pyrosequencing
  • Galaxy
  • Tissue Typing
  • Cellular Immunity
  • Multiplexing
ispartof: Lank, 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.
description: Background: 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.
language: eng
source:
identifier: ISSN: 1471-2164 ; DOI: 10.1186/1471-2164-13-378
fulltext: fulltext_linktorsrc
issn:
  • 1471-2164
  • 14712164
url: Link


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titleUltra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing
creatorLank, Simon M ; Golbach, Brittney A ; Creager, Hannah M ; Wiseman, Roger W ; Keskin, Derin Benerci ; Reinherz, Ellis Leonard ; Brusic, Vladimir ; O’connor, David H
ispartofLank, 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.
identifierISSN: 1471-2164 ; DOI: 10.1186/1471-2164-13-378
subjectHla ; Genotyping ; Roche/454 ; Pyrosequencing ; Galaxy ; Tissue Typing ; Cellular Immunity ; Multiplexing
descriptionBackground: 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.
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titleUltra-high resolution HLA genotyping and allele discovery by highly multiplexed cDNA amplicon pyrosequencing
descriptionBackground: 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.
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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.
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