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Related Articles Overcoming methodical limits of standard RHD genotyping by next-generation sequencing.
Vox Sang. 2010 Dec 7;
Authors: Stabentheiner S, Danzer M, Niklas N, Atzmüller S, Pröll J, Hackl C, Polin H, Hofer K, Gabriel C
Background and objectives Molecular variations of the RHD gene may result in the reduced expression of the D antigen and altered Rh phenotypes. In many occasions, they cannot be typed reliably by standard serological methods. Sequence-based typing is the gold standard to determine rare and unknown RHD genotypes. For this pilot study, sequence-based typing by standard Sanger sequencing was compared to a newly established next-generation sequencing approach based on pyrosequencing. Materials and methods Twenty-six DNA samples were selected after primary serological testing exhibiting a weak reaction in Rh phenotype. Parallel sequence analysis of the complete coding sequence including adjacent intronic sequences allowed a comparison of the methodical potency in mutation detection of Sanger with next-generation sequencing. Results Sanger sequencing revealed 39 RHD polymorphisms in 21 of 26 samples in the RHD coding region, while pyrosequencing detected all but two alterations resulting in a concordance rate of 94·9% and clearly revealed a heterozygous compound mutation in one sample with RHD? and Weak D type 4 alleles. The resolution of cis/trans linkage of polymorphisms and exact characterization of a 37 bp duplication was achieved by next-generation sequencing. Conclusion Our data suggest that next-generation sequencing offers a new development for high-throughput and clonal sequencing for molecular RHD genotyping. However, further attempts in the methodical set-up have to be undertaken prior to validation and introduction as a routine service.
PMID: 21133932 [PubMed - as supplied by publisher]
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Related Articles Overcoming methodical limits of standard RHD genotyping by next-generation sequencing.
Vox Sang. 2010 Dec 7;
Authors: Stabentheiner S, Danzer M, Niklas N, Atzmüller S, Pröll J, Hackl C, Polin H, Hofer K, Gabriel C
Background and objectives Molecular variations of the RHD gene may result in the reduced expression of the D antigen and altered Rh phenotypes. In many occasions, they cannot be typed reliably by standard serological methods. Sequence-based typing is the gold standard to determine rare and unknown RHD genotypes. For this pilot study, sequence-based typing by standard Sanger sequencing was compared to a newly established next-generation sequencing approach based on pyrosequencing. Materials and methods Twenty-six DNA samples were selected after primary serological testing exhibiting a weak reaction in Rh phenotype. Parallel sequence analysis of the complete coding sequence including adjacent intronic sequences allowed a comparison of the methodical potency in mutation detection of Sanger with next-generation sequencing. Results Sanger sequencing revealed 39 RHD polymorphisms in 21 of 26 samples in the RHD coding region, while pyrosequencing detected all but two alterations resulting in a concordance rate of 94·9% and clearly revealed a heterozygous compound mutation in one sample with RHD? and Weak D type 4 alleles. The resolution of cis/trans linkage of polymorphisms and exact characterization of a 37 bp duplication was achieved by next-generation sequencing. Conclusion Our data suggest that next-generation sequencing offers a new development for high-throughput and clonal sequencing for molecular RHD genotyping. However, further attempts in the methodical set-up have to be undertaken prior to validation and introduction as a routine service.
PMID: 21133932 [PubMed - as supplied by publisher]
More...