title: Next-generation sequencing is a credible strategy for blood group genotyping.
abstract: Although several medium/high-throughput tools have been engineered for molecular analysis of blood group genes, they usually rely on the targeting of single nucleotide polymorphisms, while other variants remain unidentified. To circumvent this limitation a strategy for genotyping blood group genes by next-generation sequencing (NGS) was set up. Libraries consisting of exons, flanking introns and untranslated regions of 18 genes involved in 15 blood systems were generated by the Ion AmpliSeq(™) Library Kit 2.0 and by fragmenting polymerase chain reaction products, normalized by two different approaches, mixed and sequenced by the Ion Torrent Personal Genome Machine (PGM(™) ) Sequencer. In our conditions, defined to limit both intra- and inter-sample variability, sequences from mixed libraries were read in a single run for a total coverage of 86·03% of the coding DNA sequences, including all loci defining the most clinically relevant antigens in all genes, except ABO. Importantly, the challenging attempt to generate gene-specific data for the homologous genes was successful. This work, which combines two complementary approaches to generate libraries, defines technical conditions for genotyping blood group genes, illustrates that NGS is suitable for such an application and suggests that, after automation, this novel tool could be used for molecular typing at the laboratory level.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM), U1078, Brest, France; Etablissement Français du Sang (EFS) - Bretagne, Brest, France.
authors.forename: Yann
authors.initials: Y
authors.lastname: Fichou
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Audrézet
authors.forename: Paul
authors.initials: P
authors.lastname: Guéguen
authors.forename: Cédric
authors.initials: C
authors.lastname: Le Maréchal
authors.forename: Claude
authors.initials: C
authors.lastname: Férec
mesh_terms: D001789:Blood Group Antigens / Q000235:genetics*; D001788:Blood Grouping and Crossmatching / Q000379:methods*; D005260:Female; D056426:Genetic Loci; D060005:Genotyping Techniques / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008297:Male; D011933:Reagent Kits, Diagnostic
chemical_list: D001789:Blood Group Antigens; D011933:Reagent Kits, Diagnostic
keywords: blood groups; genotyping; homologous genes; library; next-generation sequencing