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Updated: Jun 6, 2026
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Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease
Published on: April 4, 2018
Overcoming methodical limits of standard RHD genotyping by next-generation sequencing
S Stabentheiner1, M Danzer, N Niklas
1Red Cross Transfusion Service for Upper Austria, Krankenhausstrasse 7, Linz, Austria. stephanie.stabentheiner@o.roteskreuz.at
Vox Sanguinis
|December 8, 2010
Summary
Next-generation sequencing, specifically pyrosequencing, offers a powerful new method for RHD genotyping, accurately detecting RHD gene variations missed by standard Sanger sequencing. This advancement improves the molecular characterization of complex RhD phenotypes.
Area of Science:
- Genetics
- Molecular Biology
- Immunology
Background:
- Molecular variations in the RHD gene can lead to reduced D antigen expression and altered Rh phenotypes, often challenging standard serological typing.
- Sequence-based typing is the definitive method for identifying rare and unknown RHD genotypes.
Purpose of the Study:
- To compare the efficacy of standard Sanger sequencing with a novel next-generation sequencing (NGS) pyrosequencing approach for RHD genotyping.
- To evaluate the mutation detection capabilities of both sequencing methods in samples with weak RhD phenotypes.
Main Methods:
- Twenty-six DNA samples with weak RhD reactions were analyzed using both Sanger sequencing and NGS pyrosequencing.
- Sequence analysis covered the complete coding region and adjacent intronic sequences of the RHD gene.
Main Results:
- Sanger sequencing identified 39 RHD polymorphisms in 21 samples.
- NGS pyrosequencing detected all but two alterations, achieving a 94.9% concordance rate and resolving complex mutations and duplications.
- NGS provided superior resolution for cis/trans linkage and characterization of a 37 bp duplication.
Conclusions:
- NGS pyrosequencing represents a significant advancement for high-throughput, clonal molecular RHD genotyping.
- Further methodological refinement is necessary before NGS can be implemented as a routine diagnostic service.
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